Lipolytic enzyme uses thereof in the food industry

ABSTRACT

The invention encompasses the use of a lipolytic enzyme obtainable from one of the following genera:  Streptomyces, Corynebacterium  and  Thermobifida  in various methods and uses, wherein the lipolytic enzyme hydrolyzes a glycolipid or a phospholipid or transfers an acyl group from a glycolipid or phospholipids to an acyl acceptor. The present invention also relates to a lipolytic enzyme that hydrolyzes at least a galactolipid or transfers an acyl group from a galactolipid to one or more acyl acceptor substrates, wherein the enzyme is obtainable from  Corynebacterium  species.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/623,607 filed Jan. 16, 2007, which is a continuation-in-part ofInternational Patent Application PCT/IB2005/002602 filed Jul. 18, 2005and published as WO 2006/008653 on Jan. 26, 2006, which claims priorityfrom Great Britain Patent Application Nos. 0513859.9 filed Jul. 7, 2005and 0416035.4 filed Jul. 16, 2004, and from U.S. Patent Application No.60/591,185 filed Jul. 26, 2004.

Each of the above referenced applications, and each document cited inthis text (“application cited documents”) and each document cited orreferenced in each of the application cited documents, and anymanufacturer's specifications or instructions for any products mentionedin this text and in any document incorporated into this text, are herebyincorporated herein by reference; and, technology in each of thedocuments incorporated herein by reference can be used in the practiceof this invention.

It is noted that in this disclosure, terms such as “comprises”,“comprised”, “comprising”, “contains”, “containing” and the like canhave the meaning attributed to them in U.S. Patent law; e.g., they canmean “includes”, “included”, “including” and the like. Terms such as“consisting essentially of” and “consists essentially of” have themeaning attributed to them in U.S. Patent law, e.g., they allow for theinclusion of additional ingredients or steps that do not detract fromthe novel or basic characteristics of the invention, i.e., they excludeadditional unrecited ingredients or steps that detract from novel orbasic characteristics of the invention, and they exclude ingredients orsteps of the prior art, such as documents in the art that are citedherein or are incorporated by reference herein, especially as it is agoal of this document to define embodiments that are patentable, e.g.,novel, nonobvious, inventive, over the prior art, e.g., over documentscited herein or incorporated by reference herein. And, the terms“consists of” and “consisting of” have the meaning ascribed to them inU.S. Patent law; namely, that these terms are closed ended.

FIELD OF INVENTION

The present invention relates to a novel lipolytic enzyme, in particulara novel lipolytic enzyme, and nucleotide sequences encoding same. Thepresent invention also relates to methods of production of the novellipolytic enzyme and to uses thereof. The present invention also relatesto methods and uses of a lipolytic enzyme.

TECHNICAL BACKGROUND

The beneficial use of lipolytic enzymes active on glycolipids in breadmaking was taught in EP 1 193 314. It was taught that the partialhydrolysis products the lyso-glycolipids were found to have very highemulsifier functionality. However, the enzymes taught in EP 1 193 314were also found to have significant non-selective activity ontriglycerides which resulted in unnecessarily high free fatty acid.

A lipolytic enzyme from Fusarium oxysporum having phospholipase activityhas been taught in EP 0 869 167. This lipolytic enzyme has hightriacylglyceride hydrolysing (lipase) activity. This enzyme is now soldby Novozymes A/S (Denmark) as Lipopan F™.

WO02/00852 discloses five lipase enzymes and their encodingpolynucleotides, isolated from Fusarium venenatum, F. sulphureum,Aspergillus berkeleyanum, F. culmorum and F. solani. All five enzymesare described as having triacylglycerol hydrolysing activity,phospholipase and galactolipase activity.

Lipolytic enzyme variants, with specific amino acid substitutions andfusions, have been produced; some of which have an enhanced activity onthe polar lipids compared to the wildtype parent enzymes. WO01/39602describes such a variant, referred to as SP979, which is a fusion of theThermomyces lanuginosus lipase, and the Fusarium oxysporum lipasedescribed in EP 0 869 167. This variant has been found to have asignificantly high ratio of activity on phospholipids and glycolipidscompared to triglycerides.

In WO02/094123 it was discovered that by selecting lipolytic enzymeswhich were active on the polar lipids (glycolipids and phospholipids) ina dough, but substantially not active on triglycerides or1-mono-glycerides an improved functionality could be achieved.

In co-pending PCT application number PCT/IB2005/000875, wild-typelipolytic enzymes having a higher ratio of activity on polar lipids ascompared with triglycerides are taught. However, this document does notteach lipolytic enzymes from Streptomyces, Thermobifida orCorynebacterium species.

Prior to the present invention no lipolytic enzymes having activity orsignificant activity on glycolipids had been published from Streptomycesspecies. Likewise, no lipolytic enzymes having activity or significantactivity on glycolipids had been published from Thermobifida species orCorynebacterium species. Although lipases, i.e. triacylglycerolhydrolysing enzymes, have been isolated from Streptomyces species (seeVujaklija et al Arch Microbiol (2002) 178: 124-130 for example), theseenzymes have never been identified as having glycolipid hydrolysingactivity.

ASPECTS OF THE INVENTION

The present invention in predicated upon the seminal finding of alipolytic enzyme having significant galactolipid activity from the genusStreptomyces. In particular the lipolytic enzyme from the genusStreptomyces has significant galactolipid hydrolysing activity and/orsignificant galactolipid acyltransferase activity, particularly whenused in the methods and uses according to the present invention.

In addition, the present invention in predicated upon the seminalfinding that lipolytic enzymes from the genera Thermobifida orCorynebacterium have significant galactolipid activity. In particularthe lipolytic enzymes from the genera Thermobifida or Corynebacteriumhave significant galactolipid hydrolysing activity and/or significantgalactolipid acyltransferase activity, particularly when used in themethods and uses of the present invention.

In a broad aspect the present invention relates to a lipolytic enzymecapable of hydrolysing at least glycolipids and/or capable oftransferring an acyl group from at least a glycolipid to one or moreacyl acceptor substrates, wherein the enzyme is obtainable, preferablyobtained, from Streptomyces species.

In a further aspect the present invention relates to a lipolytic enzymecapable of hydrolysing at least galactolipids and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptor substrates, wherein the enzyme is encoded by a nucleicacid selected from the group consisting of:

-   a) a nucleic acid comprising a nucleotide sequence shown in SEQ ID    No. 3;-   b) a nucleic acid which is related to the nucleotide sequence of SEQ    ID No. 3 by the degeneration of the genetic code; and-   c) a nucleic acid comprising a nucleotide sequence which has at    least 70% identity with the nucleotide sequence shown in SEQ ID No.    3.

The present invention yet further provides a lipolytic enzyme comprisingan amino acid sequence as shown in SEQ ID No. 4 or an amino acidsequence which has at least 60% identity thereto.

In another aspect the present invention provides a lipolytic enzymecapable of hydrolysing at least a galactolipid and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptor substrates, wherein the enzyme comprises an amino acidsequence as shown in SEQ ID No. 4 or an amino acid sequence which has atleast 60% identity thereto.

In a further aspect the present invention provides a nucleic acidencoding a lipolytic enzyme comprising an amino acid sequence as shownin SEQ ID No. 4 or an amino acid sequence which has at least 60%identity therewith.

SEQ ID No. 3 is shown in FIG. 3 and SEQ ID No. 4 is shown in FIG. 4.

The present invention yet further provides a nucleic acid encoding alipolytic enzyme, which nucleic acid is selected from the groupconsisting of:

-   a) a nucleic acid comprising a nucleotide sequence shown in SEQ ID    No. 3;-   b) a nucleic acid which is related to the nucleotide sequence of SEQ    ID No. 3 by the degeneration of the genetic code; and-   c) a nucleic acid comprising a nucleotide sequence which has at    least 70% identity with the nucleotide sequence shown in SEQ ID No.    3.

The present invention yet further provides the use of a lipolytic enzymeaccording to the present invention in a substrate (preferably afoodstuff) for preparing a lyso-glycolipid, for example digalactosylmonoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG) by treatmentof a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monogalactosyldiglyceride (MGDG)) with the lipolytic enzyme according to the presentinvention to produce the partial hydrolysis product, i.e. thelyso-glycolipid.

In a further aspect, the present invention provides the use of alipolytic enzyme according to the present invention in a substrate(preferably a foodstuff) for preparing a lyso-phospholipid, for examplelysolecithin, by treatment of a phospholipid (e.g. lecithin) with theenzyme according to the present invention to produce a partialhydrolysis product, i.e. a lyso-phospholipid.

In one broad aspect the present invention relates to a method ofpreparing a foodstuff the method comprising admixing a lipolytic enzymeof the present invention with one or more ingredients of the foodstuff.

Another broad aspect of the present invention relates to a method ofpreparing a baked product from a dough, the method comprising admixing alipolytic enzyme of the present invention with the dough.

In a further aspect the present invention relates to a method ofpreparing a dairy product, the method comprising admixing a lipolyticenzyme of the present invention with one or more ingredients of thedairy product.

In another aspect the present invention relates to the use of alipolytic enzyme of the present invention in the manufacture of a dairyproduct to reduce one or more of the following detrimental effects:off-odours and/or off-flavours and/or soapy taste.

In another aspect of the present invention there is provided the use ofa lipolytic enzyme according to the present invention in a process oftreating egg or egg-based products to produce lysophospholipids.

In another aspect of the present invention there is provided the use ofa lipolytic enzyme according to the present invention in a process oftreating egg or egg-based products to produce lysoglycolipids.

A further aspect of the present invention provides a process ofenzymatic degumming of vegetable or edible oils, comprising treating theedible or vegetable oil with a lipolytic enzyme according to the presentinvention so as to hydrolyse a major part of the polar lipids (e.g.phospholipid and/or glycolipid).

In another aspect the present invention provides the use of a lipolyticenzyme according to the present invention in a process comprisingtreatment of a phospholipid so as to hydrolyse fatty acyl groups.

In another aspect the present invention provides the use of a lipolyticenzyme according to the present invention in a process for reducing thecontent of a phospholipid in an edible oil, comprising treating the oilwith the lipolytic enzyme according to the present invention so as tohydrolyse a major part of the phospholipid, and separating an aqueousphase containing the hydrolysed phospholipid from the oil.

There is also provided a method of preparing a lipolytic enzymeaccording to the present invention, the method comprising transforming ahost cell with a recombinant nucleic acid comprising a nucleotidesequence coding for the lipolytic enzyme, the host cell being capable ofexpressing the nucleotide sequence coding for the polypeptide of thelipolytic enzyme, cultivating the transformed host cell under conditionswhere the nucleic acid is expressed and harvesting the lipolytic enzyme.

In a further aspect the present invention relates to the use of alipolytic enzyme in accordance with the present invention in thebioconversion of polar lipids (preferably glycolipids) to make highvalue products, such as carbohydrate esters and/or protein esters and/orprotein subunit esters and/or a hydroxy acid ester.

Another aspect of the present invention relates to the use of alipolytic enzyme in accordance with the present invention in a processof enzymatic degumming of vegetable or edible oil, comprising treatingsaid edible or vegetable oil with said lipolytic enzyme so as tohydrolyse a major part of the polar lipids.

A further aspect of the present invention relates to the use of alipolytic enzyme in accordance with the present invention in a processcomprising treatment of a phospholipid so as to hydrolyse fatty acylgroups.

The present invention yet further relates to an immobilised lipolyticenzyme in accordance with the present invention.

Another aspect of the present invention relates to a method of preparinga lysoglycolipid comprising treating a substrate comprising a glycolipidwith at least one lipolytic enzyme to produce said lysoglycolipid,wherein said lipolytic enzyme has glycolipase activity and wherein saidlipolytic enzyme is obtainable from one of the following genera:Streptomyces, Corynebacterium and Thermobifida.

A further aspect of the present invention relates to a method ofpreparing a lysophospholipid comprising treating a substrate comprisinga phospholipid with at least one lipolytic enzyme to produce saidlysophospholipid, wherein said lipolytic enzyme has phospholipaseactivity and wherein said lipolytic enzyme is obtainable from one of thefollowing genera: Streptomyces, Corynebacterium and Thermobifida.

Another aspect of the present invention relates to a method of enzymaticdegumming of vegetable or edible oil, comprising treating said edible orvegetable oil with a lipolytic enzyme obtainable from one of thefollowing genera: Streptomyces, Corynebacterium and Thermobifida capableof hydrolysing a major part of the polar lipids.

The present invention further relates to a method of bioconversion ofpolar lipids to make high value products comprising treating said polarlipids with a lipolytic enzyme obtainable from one of the followinggenera: Streptomyces, Corynebacterium and Thermobifida to produce saidhigh value products, wherein said lipolytic enzyme is capable ofhydrolysing said polar lipids.

Another aspect of the present invention relates to a method of preparinga foodstuff comprising admixing at least one lipolytic enzyme with oneor more ingredients of a foodstuff wherein said lipolytic enzyme iscapable of hydrolysing a glycolipid and/or a phospholipid present in oras at least one of said ingredients and wherein said lipolytic enzyme isobtainable from one of the following genera: Streptomyces,Corynebacterium and Thermobifida.

A further aspect of the present invention relates the use of a lipolyticenzyme in a substrate for preparing a lysophospholipid wherein saidlipolytic enzyme has phospholipase activity and wherein said lipolyticenzyme is obtainable from one of the following: Streptomyces,Corynebacterium and Thermobifida.

The present invention additionally relates to the use of a lipolyticenzyme obtainable from one of the following genera: Streptomyces,Corynebacterium and Thermobifida for enzymatic degumming of vegetable oredible oil so as to hydrolyse a major part of the polar lipids.

Another aspect of the present invention relates to the use of alipolytic enzyme obtainable from one of the following genera:Streptomyces, Corynebacterium and Thermobifida in a process comprisingtreatment of a phospholipid so as to hydrolyse fatty acyl groups.

A further aspect of the present invention relates to use of a lipolyticenzyme in the bioconversion of polar lipids to make high value products,wherein said lipolytic enzyme is capable of hydrolysing said polarlipids and wherein said lipolytic enzymes is obtainable from one of thefollowing genera: Streptomyces, Corynebacterium and Thermobifida.

A further aspect of the present invention relates to the use of alipolytic enzyme obtainable from one of the following genera:Streptomyces, Corynebacterium and Thermobifida in the preparation of afoodstuff, wherein said lipolytic enzyme is capable of hydrolysing aglycolipid and/or a phospholipid.

Aspects of the present invention are presented in the claims and in thefollowing commentary.

Other aspects concerning the nucleotide sequences which can be used inthe present invention include: a construct comprising the sequences ofthe present invention; a vector comprising the sequences for use in thepresent invention; a plasmid comprising the sequences for use in thepresent invention; a transformed cell comprising the sequences for usein the present invention; a transformed tissue comprising the sequencesfor use in the present invention; a transformed organ comprising thesequences for use in the present invention; a transformed hostcomprising the sequences for use in the present invention; a transformedorganism comprising the sequences for use in the present invention. Thepresent invention also encompasses methods of expressing the nucleotidesequence for use in the present invention using the same, such asexpression in a host cell; including methods for transferring same.

The present invention further encompasses methods of isolating thenucleotide sequence, such as isolating from a host cell.

Other aspects concerning the amino acid sequence for use in the presentinvention include: a construct encoding the amino acid sequences for usein the present invention; a vector encoding the amino acid sequences foruse in the present invention; a plasmid encoding the amino acidsequences for use in the present invention; a transformed cellexpressing the amino acid sequences for use in the present invention; atransformed tissue expressing the amino acid sequences for use in thepresent invention; a transformed organ expressing the amino acidsequences for use in the present invention; a transformed hostexpressing the amino acid sequences for use in the present invention; atransformed organism expressing the amino acid sequences for use in thepresent invention. The present invention also encompasses methods ofpurifying the amino acid sequence for use in the present invention usingthe same, such as expression in a host cell; including methods oftransferring same, and then purifying said sequence.

For the ease of reference, these and further aspects of the presentinvention are now discussed under appropriate section headings. However,the teachings under each section are not necessarily limited to eachparticular section.

DETAILED DISCLOSURE OF THE INVENTION

Suitably, the lipolytic enzyme for use in the methods and uses accordingto the present invention may be a lipolytic enzyme comprising any one ofthe amino acid sequences shown as SEQ ID No. 4, 5, 7, 8, 12, 14 or 16 oran amino acid sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97% or 98% identity therewith, or encoded by any one of thenucleotide sequences shown as SEQ ID No. 3, 6, 9, 13, 15 or 17 or anucleotide sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97% or 98% identity therewith.

Preferably, the lipolytic enzyme for use in the methods and usesaccording to the present invention is a lipolytic enzyme capable ofhydrolysing at least galactolipids and/or capable of transferring anacyl group from at least a galactolipid to one or more acyl acceptorsubstrates, wherein the enzyme is obtainable, preferably obtained, fromStreptomyces species.

In one embodiment the lipolytic enzyme for use in the methods and usesaccording to the present invention is preferably a lipolytic enzymecapable of hydrolysing at least galactolipids and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptor substrates, wherein the enzyme is encoded by a nucleicacid selected from the group consisting of:

-   -   a) a nucleic acid comprising a nucleotide sequence shown in SEQ        ID No. 3;    -   b) a nucleic acid which is related to the nucleotide sequence of        SEQ ID No. 3 by the degeneration of the genetic code; and    -   c) a nucleic acid comprising a nucleotide sequence which has at        least 70% identity with the nucleotide sequence shown in SEQ ID        No. 3.

In one embodiment, the lipolytic enzyme for use in the methods and usesaccording to the present invention is preferably a lipolytic enzymecomprising an amino acid sequence as shown in SEQ ID No. 4 or an aminoacid sequence which has at least 60% identity thereto.

In another embodiment the lipolytic enzyme for use in the methods anduses according to the present invention is preferably a lipolytic enzymecapable of hydrolysing at least a galactolipid and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptor substrates, wherein the enzyme comprises an amino acidsequence as shown in SEQ ID No. 4 or an amino acid sequence which has atleast 60% identity thereto.

Preferably, the lipolytic enzyme for use in the methods and usesaccording to the present invention is a lipolytic enzyme capable ofhydrolysing at least galactolipids and/or capable of transferring anacyl group from at least a galactolipid to one or more acyl acceptorsubstrates, wherein the enzyme is obtainable, preferably obtained, fromThermobifida species, preferably Thermobifida fusca.

Preferably, the lipolytic enzyme for use in the methods and usesaccording to the present invention is a lipolytic enzyme capable ofhydrolysing at least galactolipids and/or capable of transferring anacyl group from at least a galactolipid to one or more acyl acceptorsubstrates, wherein the enzyme is obtainable, preferably obtained, fromCorynebacterium species, preferably Corynebacterium efficiens.

In a further embodiment the lipolytic enzyme for use in the methods anduses according to the present invention may be a lipolytic enzymecomprising any one of the amino acid sequences shown as SEQ ID No. 4, 5,7, 8, 12, 14 or 16 or an amino acid sequence which has at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identity therewith, or encodedby any one of the nucleotide sequences shown as SEQ ID No. 3, 6, 9, 13,15 or 17 or a nucleotide sequence which has at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97% or 98% identity therewith.

In a further embodiment the lipolytic enzyme for use in the methods anduses according to the present invention may be a lipolytic enzymecomprising any one of amino sequences shown as SEQ ID No. 5, 7, 8, 14 or16 or an amino acid sequence which has at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97% or 98% identity therewith for the uses described herein.

In a further embodiment the lipolytic enzyme for use in the methods anduses according to the present invention may be a lipolytic enzymecomprising any one of amino sequences shown as SEQ ID No. 5, 7 or 16 oran amino acid sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97% or 98% identity therewith for the uses described herein.

More preferably in one embodiment the lipolytic enzyme for use in themethods and uses according to the present invention may be a lipolyticenzyme comprising the amino acid sequence shown as SEQ ID No. 16 or anamino acid sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97% or 98% identity therewith.

In another embodiment the lipolytic enzyme for use in the methods anduses according to the present invention may be a lipolytic enzymecomprising the amino acid sequence shown as SEQ ID Nos. 12 or 14 or anamino acid sequence which has at least 80%, 85%, 90%, 95%, 96%, 97% or98% identity therewith.

In another embodiment the lipolytic enzyme for use in the methods anduses according to the present invention may be a lipolytic enzymecomprising the amino acid sequence shown as SEQ ID No. 8 or an aminoacid sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%or 98% identity therewith.

In one embodiment the lipolytic enzyme for use in the methods and usesaccording to the present invention may be a lipolytic enzyme capable ofhydrolysing at least galactolipids and/or capable of transferring anacyl group from at least a galactolipid to one or more acyl acceptorsubstrates, wherein the enzyme is encoded by a nucleic acid selectedfrom the group consisting of:

-   -   a) a nucleic acid comprising a nucleotide sequence shown in SEQ        ID No. 3;    -   b) a nucleic acid which is related to the nucleotide sequence of        SEQ ID No. 3 by the degeneration of the genetic code; and    -   c) a nucleic acid comprising a nucleotide sequence which has at        least 70% identity with the nucleotide sequence shown in SEQ ID        No. 3.

In one embodiment the lipolytic enzyme according to the presentinvention may be a lipolytic enzyme obtainable, preferably obtained,from the Streptomyces strains L130 or L131 deposited by Danisco A/S ofLangebrogade 1, DK-1001 Copenhagen K, Denmark under the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe purposes of Patent Procedure at the National Collection ofIndustrial, Marine and Food Bacteria (NCIMB) 23 St. Machar Street,Aberdeen Scotland, GB on 25 Jun. 2004 under accession numbers NCIMB41226 and NCIMB 41227, respectively.

Preferably, the lipolytic enzyme according to the present invention actson at least a glycolipid, such as digalactosyldiglyceride (DGDG) forexample. Suitably, the lipolytic enzyme according to the presentinvention may also act on one or more other polar lipid substrates, suchas a phospholipid, for example a lecithin, e.g. phosphatidylcholine.

An alternative way of expressing the term “capable of hydrolysingglycolipids” as used herein would be to say that the lipolytic enzymehas glycolipid hydrolysing activity.

Preferably, the lipolytic enzyme according to the present inventionhydrolyses a glycolipid, such as digalactosyldiglyceride (DGDG) forexample, and also a phospholipid, such as a lecithin, e.g.phosphatidylcholine.

Preferably the lipolytic enzyme according to the present invention actson glycolipids such as DGDG or MGDG.

In one aspect the lipolytic enzyme according to the present inventionhydrolyses DGDG to DGMG and/or MGDG to MGMG.

In one aspect the lipolytic enzyme according to the present inventionhydrolyses lecithin to lysolecithin.

When it is the case that the lipolytic enzyme is capable of transferringan acyl group from at least a glycolipid to a donor substrate, the polarlipid substrate may be referred to herein as the “lipid acyl donor”.

In one embodiment, the enzyme according to the present invention whichas well as having phospholipase and/or glycolipase activity (generallyclassified as E.C. 3.1.1.26; E.C. 3.1.1.4 or E.C. 3.1.1.32 in accordancewith the Enzyme Nomenclature Recommendations (1992) of the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology) also has acyltransferase activity (generally classified as E.C.2.3.1.x), whereby the enzyme is capable of transferring an acyl groupfrom a lipid acyl donor to one or more acceptor substrates, such as oneor more of the following: a sterol; a stanol; a carbohydrate; a protein;a protein subunit; glycerol.

Lipid acyltransferases and their uses are taught in co-pendingInternational Patent Application number PCT/IB2004/000655. This documentis incorporated herein by reference. However, the lipolytic enzymes fromthe genera Streptomyces according to the present invention are nottaught in PCT/IB2004/000655.

In some aspects, the lipolytic enzyme for use in the methods and/or usesof the present invention may be capable of transferring an acyl groupfrom a polar lipid (as defined herein) to one or more of the followingacyl acceptor substrates: a sterol, a stanol, a carbohydrate, a proteinor subunits thereof, or a glycerol.

For some aspects the “acyl acceptor” according to the present inventionmay be any compound comprising a hydroxy group (—OH), such as forexample, polyvalent alcohols, including glycerol; sterol; stanols;carbohydrates; hydroxy acids including fruit acids, citric acid,tartaric acid, lactic acid and ascorbic acid; proteins or a sub-unitthereof, such as amino acids, protein hydrolysates and peptides (partlyhydrolysed protein) for example; and mixtures and derivatives thereof.

In some aspects, the “acyl acceptor” according to the present inventionmay be preferably not water.

In one embodiment, the acyl acceptor is preferably not a monoglycerideand/or a diglyceride.

In one aspect, preferably the enzyme is capable of transferring an acylgroup from a lipid to a sterol and/or a stanol.

In one aspect, preferably the enzyme is capable of transferring an acylgroup from a lipid to a carbohydrate.

In one aspect, preferably the enzyme is capable of transferring an acylgroup from a lipid to a protein or a subunit thereof. Suitably theprotein subunit may be one or more of the following: an amino acid, aprotein hydrolysate, a peptide, a dipeptide, an oligopeptide, apolypeptide.

Suitably in the protein or protein subunit the acyl acceptor may be oneor more of the following constituents of the protein or protein subunit:a serine, a threonine, a tyrosine, or a cysteine.

When the protein subunit is an amino acid, suitably the amino acid maybe any suitable amino acid. Suitably the amino acid may be one or moreof a serine, a threonine, a tyrosine, or a cysteine for example.

In one aspect, preferably the enzyme is capable of transferring an acylgroup from a lipid to glycerol.

In one aspect, preferably the enzyme is capable of transferring an acylgroup from a lipid to a hydroxy acid.

In one aspect, preferably the enzyme is capable of transferring an acylgroup from a lipid to a polyvalent alcohol.

In one aspect, the lipolytic enzyme may, as well as being able totransfer an acyl group from a lipid to a sterol and/or a stanol,additionally be able to transfer the acyl group from a lipid to one ormore of the following: a carbohydrate, a protein, a protein subunit,glycerol.

The term lecithin as used herein encompasses phosphatidylcholine,phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine andphosphatidylglycerol.

For some aspects, preferably the lipid substrate is at least aglycolipid, such as DGDG for example.

For some aspects, preferably the lipid substrate may be additionally aphospholipid, such as lecithin, for example phosphatidylcholine. Otherphospholipid substrates in accordance with the present invention may beone or more of N acyl phosphatidyl ethanolamine (APE) or N acyllyso-phosphatidyl ethanolamine (ALPE).

Preferably the lipid substrate is a food lipid, that is to say a lipidcomponent of a foodstuff.

For some aspects, preferably the lipolytic enzyme according to thepresent invention is incapable, or substantially incapable, of acting ona triglyceride and/or a 1-monoglyceride and/or 2-monoglyceride.

In one embodiment the lipolytic enzyme according to the presentinvention has no activity or no significant activity on triglycerideand/or 1-monoglycerides and/or 2-monoglycerides.

Suitably, the lipid substrate or lipid acyl donor may be one or morelipids present in one or more of the following substrates: fats,including lard, tallow and butter fat; oils including oils extractedfrom or derived from palm oil, sunflower oil, soya bean oil, saffloweroil, cotton seed oil, ground nut oil, corn oil, olive oil, peanut oil,coconut oil, and rapeseed oil. Lecithin from soya, rapeseed or egg yolkis also a suitable lipid substrate. The lipid substrate may be an oatlipid or other plant based material containing galactolipids.

In one aspect the lipid substrate or lipid acyl donor is preferablylecithin (such as phosphatidylcholine) in egg yolk.

For some aspects of the present invention, the lipid may be selectedfrom lipids having a fatty acid chain length of from 8 to 22 carbons.

For some aspects of the present invention, the lipid may be selectedfrom lipids having a fatty acid chain length of from 16 to 22 carbons,more preferably of from 16 to 20 carbons.

For some aspects of the present invention, the lipid may be selectedfrom lipids having a fatty acid chain length of no greater than 14carbons, suitably from lipids having a fatty acid chain length of from 4to 14 carbons, suitably 4 to 10 carbons, suitably 4 to 8 carbons.

Suitably, the lipolytic enzyme according to the present inventionexhibits at least glycolipase activity (E.C. 3.1.1.26). Suitably, thelipolytic enzyme according to the present invention may also exhibitphospholipase A2 activity (E.C. 3.1.1.4) and/or phospholipase A1activity (E.C. 3.1.1.32).

For some aspects, the lipolytic enzyme according to the presentinvention may solely have glycolipase activity (E.C. 3.1.1.26).

For some aspects, the lipolytic enzyme according to the presentinvention is a galactolipase (E.C. 3.1.1.26). The fact that the enzymeis designated at a galactolipase does not, however, prevent it fromhaving other side-activities, such as activity towards other polarlipids for example.

The terms “glycolipase activity” and “galactolipase activity” as usedherein are used interchangeably.

Suitably, for some aspects the lipolytic enzyme according to the presentinvention may be capable of transferring an acyl group from a glycolipidand/or a phospholipid to one or more acceptor substrates.

Suitably the acceptor substrate may be one or more of the followingsubstrates: a sterol, a stanol, a carbohydrate, a protein, glycerol.

The term “polar lipids” as used herein means phospholipids and/orglycolipids. In some aspects, the term polar lipids preferably means atleast glycolipids.

The glycolipase activity; phospholipase activity and/or triacylglycerollipase activity of an enzyme can be determined using the assayspresented hereinbelow.

Determination of Galactolipase Activity (Glycolipase Activity Assay(GLU-7)): Substrate

0.6% digalactosyldiglyceride (Sigma D 4651), 0.4% Triton-X 100 (SigmaX-100) and 5 mM CaCl₂ was dissolved in 0.05M HEPES buffer pH 7.

Assay Procedure:

400 μL substrate was added to an 1.5 mL Eppendorf tube and placed in anEppendorf Thermomixer at 37° C. for 5 minutes. At time t=0 min, 50 μLenzyme solution was added. Also a blank with water instead of enzyme wasanalyzed. The sample was mixed at 10×100 rpm in an Eppendorf Thermomixerat 37° C. for 10 minutes. At time t=10 min the Eppendorf tube was placedin another thermomixer at 99° C. for 10 minutes to stop the reaction.

Free fatty acid in the samples was analyzed by using the NEFA C kit fromWAKO GmbH.

Enzyme activity GLU at pH 7 was calculated as micromole fatty acidproduced per minute under assay conditions

Determination of Phospholipase Activity (Phospholipase Activity Assay(PLU-7)): Substrate

0.6% L-α Phosphatidylcholine 95% Plant (Avanti #441601), 0.4% Triton-X100 (Sigma X-100) and 5 mM CaCl₂ was dispersed in 0.05M HEPES buffer pH7.

Assay Procedure:

400 μL substrate was added to a 1.5 mL Eppendorf tube and placed in anEppendorf Thermomixer at 37° C. for 5 minutes. At time t=0 min, 50 μLenzyme solution was added. Also a blank with water instead of enzyme wasanalyzed. The sample was mixed at 10×100 rpm in an Eppendorf Thermomixerat 37° C. for 10 minutes. At time t=10 min the Eppendorf tube was placedin another thermomixer at 99° C. for 10 minutes to stop the reaction.

Free fatty acid in the samples was analyzed by using the NEFA C kit fromWAKO GmbH.

Enzyme activity PLU-7 at pH 7 was calculated as micromole fatty acidproduced per minute under assay conditions

Determination of Triacylglyceride Lipase Activity: Assay Based onTriglyceride (Tributyrin) as Substrate (LIPU):

Lipase activity based on tributyrin is measured according to FoodChemical Codex, Forth Edition, National Academy Press, 1996, p 803. Withthe modification that the sample is dissolved in deionized water instead of glycine buffer, and the pH stat set point is 5.5 instead of 7.

1 LIPU is defined as the quantity of enzyme which can liberate 1micromole butyric acid per min. under assay conditions.

In one embodiment, preferably the lipolytic enzyme according to thepresent invention is a wild-type lipolytic enzyme.

The terms “natural” and “wild type” as used herein mean anaturally-occurring enzyme. That is to say an enzyme expressed from theendogenous genetic code and isolated from its endogenous host organismand/or a heterologously produced enzyme which has not been mutated (i.e.does not contain amino acid deletions, additions or substitutions) whencompared with the mature protein sequence (after co- andpost-translational cleavage events) endogenously produced. Natural andwild-type proteins of the present invention may be encoded by codonoptimised polynucleotides for heterologous expression, and may alsocomprise a non-endogenous signal peptide selected for expression in thathost.

The term “variant” as used herein means a protein expressed from anon-endogenous genetic code resulting in one or more amino acidalterations (i.e. amino acid deletions, additions or substitutions) whencompared with the natural or wild-type sequence within the matureprotein sequence.

Preferably, the lipolytic enzyme according to the present invention isobtainable (suitably may be obtained) from a bacterium.

Preferably, the lipolytic enzyme according to the present invention maybe obtainable (preferably obtained) from Streptomyces spp. Preferably,the lipolytic enzyme according to the present invention may beobtainable (preferably obtained) from Streptomyces strain L131 orStreptomyces strain L130.

Preferably, the lipolytic enzyme according to the present inventioncomprises an amino acid sequence which has at least 70%, preferably atleast 75%, preferably at least 80%, preferably at least 90%, preferablyat least 95%, preferably at least 98%, preferably at least 99% identitywith the amino acid sequence shown as SEQ ID No. 4.

Preferably, the nucleic acid encoding the lipolytic enzyme according tothe present invention comprises a nucleotide sequence which has at least75%, preferably at least 80%, preferably at least 85%, preferably atleast 90%, preferably at least 95%, preferably at least 98%, preferablyat least 99% identity with the nucleotide sequence shown in SEQ ID No.3.

In one embodiment suitably the pH optimum of the enzyme on agalactolipid substrate is about 6-8, preferably about 6.5 to 7.5, morepreferably about 7.

Suitably, the lipolytic enzyme according to the present invention maynot be inhibited or not significantly be inhibited by lipases inhibitorspresent in wheat flour. The term “not significantly inhibited” as usedherein means that the enzyme is less sensitive to lipase inhibitorspresent in the wheat flour when compared to an equivalent dosage (PLU)of LipopanF™ (Novozymes A/S, Denmark), as based on the standardphospholipase (PLU-7) assay defined herein.

Suitably, the lipolytic enzyme according to the present invention iscapable of hydrolysing at least 10% of the galactolipid diester in thesubstrate (i.e. in the foodstuff, e.g. dough, for instance) to themonoester. Preferably, the enzyme is capable of hydrolysing at least20%, more preferably at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80% or at least 90% of the galactolipiddiester to the mono ester. Suitably, the galactolipid diester may be oneor more of MGDG or DGDG and the monoester may be one or more of MGMG orDGMG, respectively.

Suitably, the lipolytic enzyme according to the present invention may beisolated from a fermentation broth of Streptomyces strain L131 orStreptomyces strain L130.

Suitably, the enzyme may be purified by liquid chromatography.

The amino acid sequence of the purified lipolytic enzyme may bedetermined by Edman degradation, LC-MS and MALDI-TOF analysis.

Suitably, the enzyme as defined herein may catalyse one or more of thefollowing reactions: interesterification, transesterification,alcoholysis, hydrolysis.

The term “interesterification” refers to the enzymatic catalysedtransfer of acyl groups between a lipid donor and lipid acceptor,wherein the lipid donor is not a free acyl group.

The term “transesterification” as used herein means the enzymaticcatalysed transfer of an acyl group from a lipid donor (other than afree fatty acid) to an acyl acceptor (other than water).

As used herein, the term “alcoholysis” refers to the enzymatic cleavageof a covalent bond of an acid derivative by reaction with an alcohol ROHso that one of the products combines with the H of the alcohol and theother product combines with the OR group of the alcohol.

As used herein, the term “alcohol” refers to an alkyl compoundcontaining a hydroxyl group.

As used herein, the term “hydrolysis” refers to the enzymatic catalysedtransfer of an acyl group from a lipid to the OH group of a watermolecule. Acyl transfer which results from hydrolysis requires theseparation of the water molecule.

The term “foodstuff” as used herein means a substance which is suitablefor human and/or animal consumption.

Suitably, the term “foodstuff” as used herein may mean a foodstuff in aform which is ready for consumption. Alternatively or in addition,however, the term foodstuff as used herein may mean one or more foodmaterials which are used in the preparation of a foodstuff. By way ofexample only, the term foodstuff encompasses both baked goods producedfrom dough as well as the dough used in the preparation of said bakedgoods.

In a preferred aspect the present invention provides a foodstuff asdefined above wherein the foodstuff is selected from one or more of thefollowing: eggs, egg-based products, including but not limited tomayonnaise, salad dressings, sauces, ice creams, egg powder, modifiedegg yolk and products made therefrom; baked goods, including breads,cakes, sweet dough products, laminated doughs, liquid batters, muffins,doughnuts, biscuits, crackers and cookies; confectionery, includingchocolate, candies, caramels, halawa, gums, including sugar free andsugar sweetened gums, bubble gum, soft bubble gum, chewing gum andpuddings; frozen products including sorbets, preferably frozen dairyproducts, including ice cream and ice milk; dairy products, includingcheese, butter, milk, coffee cream, whipped cream, custard cream, milkdrinks and yoghurts; mousses, whipped vegetable creams, meat products,including processed meat products; edible oils and fats, aerated andnon-aerated whipped products, oil-in-water emulsions, water-in-oilemulsions, margarine, shortening and spreads including low fat and verylow fat spreads; dressings, mayonnaise, dips, cream based sauces, creambased soups, beverages, spice emulsions and sauces.

Suitably the foodstuff in accordance with the present invention may be a“fine foods”, including cakes, pastry, confectionery, chocolates, fudgeand the like.

In one aspect the foodstuff in accordance with the present invention maybe a dough product or a baked product, such as a bread, a fried product,a snack, cakes, pies, brownies, cookies, noodles, snack items such ascrackers, graham crackers, pretzels, and potato chips, and pasta.

In a further aspect, the foodstuff in accordance with the presentinvention may be a plant derived food product such as flours, pre-mixes,oils, fats, cocoa butter, coffee whitener, salad dressings, margarine,spreads, peanut butter, shortenings, ice cream, cooking oils.

In another aspect, the foodstuff in accordance with the presentinvention may be a dairy product, including butter, milk, cream, cheesesuch as natural, processed, and imitation cheeses in a variety of forms(including shredded, block, slices or grated), cream cheese, ice cream,frozen desserts, yoghurt, yoghurt drinks, butter fat, anhydrous milkfat, other dairy products. The enzyme according to the present inventionmay improve fat stability in dairy products.

It is particularly advantageous to utilise the enzyme according to thepresent invention in cheese. Thus, a lipolytic enzyme in accordance withthe present invention can advantageously be used to produce cheese. Thelipolytic enzyme catalyses the hydrolysis of phospholipids in the milkwhich contributes to increased cheese yield. Preferably the lipolyticenzyme according to the present invention may be added to milk (referredto as cheese milk) prior to or during the cheese making process.

In another aspect, the foodstuff in accordance with the presentinvention may be a food product containing animal derived ingredients,such as processed meat products, cooking oils, shortenings.

In a further aspect, the foodstuff in accordance with the presentinvention may be a beverage, a fruit, mixed fruit, a vegetable or wine.In some cases the beverage may contain up to 20 g/l of addedphytosterols.

In another aspect, the foodstuff in accordance with the presentinvention may be an animal feed. The animal feed may be enriched withphytosterol and/or phytostanols, preferably with beta-sitosterol/stanol.Suitably, the animal feed may be a poultry feed. When the foodstuff ispoultry feed, the present invention may be used to lower the cholesterolcontent of eggs produced by poultry fed on the foodstuff according tothe present invention.

In one aspect preferably the foodstuff is selected from one or more ofthe following: eggs, egg-based products, including mayonnaise, saladdressings, sauces, ice cream, egg powder, modified egg yolk and productsmade therefrom.

Preferably the foodstuff according to the present invention is a watercontaining foodstuff. Suitably the foodstuff may be comprised of 10-98%water, suitably 14-98%, suitably of 18-98% water, suitably of 20-98%,suitably of 40-98%, suitably of 50-98%, suitably of 70-98%, suitably of75-98%.

For some aspects, the foodstuff in accordance with the present inventionmay not be a pure plant derived oil, such as olive oil, sunflower oil,peanut oil, rapeseed oil for instance. For the avoidance of doubt, insome aspects of the present invention the foodstuff according to thepresent invention may comprise an oil, but the foodstuff is notprimarily composed of oil or mixtures of oil. For some aspects,preferably the foodstuff comprises less than 95% lipids, preferably lessthan 90% lipids, preferably less than 85%, preferably less than 80%lipids. Thus, for some aspects of the present invention oil may be acomponent of the foodstuff, but preferably the foodstuff is not an oilper se.

The advantages of using a lipolytic enzyme capable of transferring anacyl group in food applications is taught in patent applicationsWO2004/064987, WO2004/064537, PCT/IB2004/004374 and GB0513859.9 whichare incorporated herein by reference.

The production of free fatty acids can be detrimental to foodstuffs.Free fatty acids have been linked with off-odours and/or off-flavours infoodstuffs, as well other detrimental effects, including a soapy tastein dairy products such as cheese for instance. Suitably in someembodiments of the present invention the lipolytic enzyme is capable oftransferring the fatty acid from the lipid to an acyl acceptor, forexample a sterol and/or a stanol. Hence, the overall level of free fattyacids in the foodstuff does not increase or increases only to aninsignificant degree. Thus, a lipolytic enzyme capable of transferringan acyl group according to the present invention may provide one or moreof the following unexpected technical effects in the production ofcheese: a decrease in the oiling-off effect in cheese; an increase incheese yield; an improvement in flavour; a reduced mal-odour; a reduced“soapy” taste.

The utilisation of a lipolytic enzyme taught herein which can transferthe acyl group to a carbohydrate as well as to a sterol and/or a stanolis particularly advantageous for foodstuffs comprising eggs. Inparticular, the presence of sugars, in particular glucose, in eggs andegg products is often seen as disadvantageous. Egg yolk may comprise upto 1% glucose. In accordance with the present invention this unwantedsugar can be readily removed by “esterifying” the sugar to form a sugarester.

The presence of diglycerides in edible oils is disadvantageous. Inparticular, diglycerides in edible oils (in particular palm oil) canlead to a low quality oil. Suitably in some embodiments of the presentinvention a lipolytic enzyme taught herein is capable of transferringthe fatty acid from the lipid to an acyl acceptor which reduces thelevel of diglycerides in the oil without increasing or significantlyincreasing the level of free fatty acids.

A lipolytic enzyme taught herein is able to hydrolyse a major part ofthe phospholipids in an edible or vegetable oil. This is highlyadvantageous in the enzymatic degumming of vegetable or edible oils.Suitably in some embodiments of the present invention the lipolyticenzyme may be capable of transferring the fatty acid from the lipid toan acyl acceptor. Hence, advantageously the overall level of free fattyacids in the oil does not increase or increases only to an insignificantdegree. The production of free fatty acids can be detrimental in theedible oil. Preferably, the method according to the present inventionresults in the degumming of an edible oil wherein the accumulation offree fatty acids is reduced and/or eliminated.

The claims of the present invention are to be construed to include eachof the foodstuffs listed above.

In some of the applications mentioned herein, particularly the foodapplications, such as the bakery applications, the lipolytic enzymeaccording to the present invention may be used with one or moreconventional emulsifiers, including for example monoglycerides, diacetyltartaric acid esters of mono- and diglycerides of fatty acids, sugaresters, sodium stearoyl lactylate (SSL) and lecithins.

In addition or alternatively, the enzyme according to the presentinvention may be used with one or more other suitable food gradeenzymes. Thus, it is within the scope of the present invention that, inaddition to the lipolytic enzyme of the present invention, at least onefurther enzyme may be added to the baked product and/or the dough. Suchfurther enzymes include starch degrading enzymes such as endo- orexoamylases, pullulanases, debranching enzymes, hemicellulases includingxylanases, cellulases, oxidoreductases, e.g. glucose oxidase, pyranoseoxidase, sulfhydryl oxidase or a carbohydrate oxidase such as one whichoxidises maltose, for example hexose oxidase (HOX), lipases,phospholipases and hexose oxidase, proteases, and acyltransferases (suchas those described in PCT/IB2004/000575 for instance).

The present invention encompasses food enzyme compositions, includingbread and/or dough improving compositions comprising the enzymeaccording to the present invention, and optionally further comprisinganother enzyme, such as one or more other suitable food grade enzymes,including starch degrading enzymes such as endo- or exoamylases,pullulanases, debranching enzymes, hemicellulases including xylanases,cellulases, oxidoreductases, e.g. glucose oxidase, pyranose oxidase,sulfhydryl oxidase or a carbohydrate oxidase such as one which oxidisesmaltose, for example hexose oxidase (HOX), lipases, phospholipases andhexose oxidase, proteases and acyltransferases (such as those describedin PCT/IB2004/000575 for instance).

In some applications mentioned herein, particularly in foodapplications, such as the bakery applications, the lipolytic enzymeaccording to the present invention may be added in combination orsequentially with one or more enzyme substrates. By way of example only,the lipolytic enzyme according to the present invention may be addedtogether with one or more polar lipid substrates and/or one or more acylacceptor substrates.

In some applications mentioned herein, particularly in foodapplications, such as the bakery applications, the lipolytic enzymeaccording to the present invention may be used with one or more hydroxyacids, including for example tartaric acid, citric acid, lactic acid,succinic acid or ascorbic acid for example.

The term “improved properties” as used herein means any property whichmay be improved by the action of the lipolytic enzyme of the presentinvention. In particular, the use of the lipolytic enzyme according tothe present invention results in one or more of the followingcharacteristics: increased volume of the baked product; improved crumbstructure of the baked product; anti-staling properties in the bakedproduct; increased strength, increased stability, reduced stickinessand/or improved machinability of the dough.

The improved properties are evaluated by comparison with a dough and/ora baked product prepared without addition of the lipolytic enzymeaccording to the present invention.

The term “baked product” as used herein includes a product prepared froma dough. Examples of baked products (whether of white, light or darktype) which may advantageously produced by the present invention includeone or more of the following: bread (including white, whole-meal and ryebread), typically in the form of loaves or rolls, steam buns, Frenchbaguette-type bread, pita bread, tacos, corn tortilla, wheat tortilla,cakes, pancakes, biscuits, crisp bread, pasta, noodles and the like.

The dough in accordance with the present invention may be a leaveneddough or a dough to be subjected to leavening. The dough may be leavenedin various ways such as by adding sodium bicarbonate or the like, or byadding a suitable yeast culture such as a culture of Saccharomycescerevisiae (baker's yeast).

The present invention further relates to the use of the lipolytic enzymein accordance with the present invention to produce a pasta dough,preferably prepared from durum flour or a flour of comparable quality.

The lipolytic enzyme according to the present invention is suitable foruse in the enzymatic degumming of vegetable or edible oils. Inprocessing of vegetable or edible oil the edible or vegetable oil istreated with lipolytic enzyme according to the present invention so asto hydrolyse a major part of the polar lipids (e.g. phospholipid).Preferably, the fatty acyl groups are hydrolysed from the polar lipids.The degumming process typically results in the reduction of the contentof the polar lipids, particularly of phospholipids, in an edible oil dueto hydrolyse of a major part (i.e. more than 50%) of the polar lipid,e.g. phospholipid. Typically, the aqueous phase containing thehydrolysed polar lipid (e.g. phospholipid) is separated from the oil.Suitably, the edible or vegetable oil may initially (pre-treatment withthe enzyme according to the present invention) have a phosphorus contentof 50-250 ppm.

In one embodiment, the present invention relates to the use of thelipolytic enzyme in accordance with the present invention in thebioconversion of polar lipids (preferably glycolipids) to make highvalue products, such as carbohydrate esters and/or protein esters and/orprotein subunit esters and/or a hydroxy acid ester. The use of alipolytic enzyme, particularly a lipolytic enzyme capable oftransferring acyl groups from a polar lipid substrate (preferably aglycolipid) to a acyl acceptor, in the bioconversion of polar lipids andthe advantages thereof is detailed in PCT/IB2004/004374 incorporatedherein by reference.

In one embodiment the lipolytic enzyme for use in the methods of thepresent invention may be immobilised. When it is the case that theenzyme is immobilised the admixture comprising an acyl donor, optionallyan acyl acceptor, and optionally water may be passed through a columnfor example comprising the immobilised enzyme. By immobilising theenzyme it is possible to easily reuse it.

Suitably, the immobilised enzyme may be used in a flow reactor or in abatch reactor containing a reaction mixture which comprises a lipid acyldonor and optionally an acyl acceptor dissolved in water. When the acylacceptor is present the donor and acceptor are in a two-phase system oran emulsion. The reaction mixture may be optionally stirred orsonicated. Once the reaction has reached equilibrium for example, thereaction mixture and the immobilised enzyme may be separated. Suitably,the reaction product may be fractionated for example by hydrophobicinteraction chromatography, crystallisation or high vacuum distillation.

Immobilised lipid acyl transferase can be prepared using immobilisationtechniques known in the art. There are numerous methods of preparingimmobilised enzymes, which will be apparent to a person skilled in theart (for example the techniques referred to in EP 0 746 608; or BalcaoV. M. et al Enzyme Microb Technol. 1996 May 1; 18 (6):392-416; or Retzet al Chem Phys Lipids 1998 June:93 (1-2): 3-14; Bornscheuer et alTrends Biotechnol. 2002 October; 20 (10):433-7; Plou et al Biotechnology92 (2002) 55-66; Warmuth et al 1992 Bio Forum 9, 282-283; Ferrer et al2000 J. Chem. Technol. Biotechnol. 75, 1-8; or Christensen et al 1998Nachwachsende Rohstoff 10, 98-105; Petersen and Christenen 2000 AppliedBiocatalysis Harwood Academic Publishers, Amsterdam (each of which isincorporated herein by reference).

Techniques which may be used herein include covalent coupling toEupergit C, adsorption on polypropylene and silica-granulation forexample.

Lipolytic Enzymes in Accordance with the Present Invention

The lipolytic enzyme for use in accordance with the present inventionand/or the methods described herein is preferably a lipolytic enzymecapable of hydrolysing at least galactolipids and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptor substrates, wherein the enzyme is encoded by a nucleicacid selected from the group consisting of:

-   d) a nucleic acid comprising a nucleotide sequence shown in SEQ ID    No. 3;-   e) a nucleic acid which is related to the nucleotide sequence of SEQ    ID No. 3 by the degeneration of the genetic code; and-   f) a nucleic acid comprising a nucleotide sequence which has at    least 70% identity with the nucleotide sequence shown in SEQ ID No.    3.

Preferably, the lipolytic enzyme used in accordance with the presentinvention and/or in the methods described herein is a lipolytic enzymecomprising an amino acid sequence as shown in SEQ ID No. 4 or an aminoacid sequence which has at least 60% identity thereto.

However, the lipolytic enzyme for use in accordance with the presentinvention and/or in the methods of the present invention may be anylipolytic enzyme obtainable from Streptomyces species which is capableof hydrolysing at least a galactolipid and/or capable of transferring anacyl group from a galactolipid to one or more acyl acceptor substrates.

Suitable lipolytic enzymes having galactolipase activity for use inaccordance with the present invention and/or in the methods of thepresent invention may comprise any one of the following amino acidsequences and/or be encoded by the following nucleotide sequences:

Thermobifida\fusca GDSx (SEQ ID NO: 31) 548 aa

ZP_00058717 SEQ ID No. 5 1mlphpagerg evgaffallv gtpqdrrlrl echetrplrg rcgcgerrvp pltlpgdgvl 61cttsstrdae tvwrkhlqpr pdggfrphlg vgcllagqgs pgvlwcgreg crfevcrrdt 121pglsrtrngd ssppfragws lppkcgeisq sarktpavpr ysllrtdrpd gprgrfvgsg 181praatrrrlf lgipalvlvt altlvlavpt gretlwrmwc eatqdwclgv pvdsrgqpae 241dgeflllspv qaatwgnyya lgdsyssgdg ardyypgtav kggcwrsana ypelvaeayd 301faghlsflac sgqrgyamld aidevgsqld wnsphtslvt igiggndlgf stvlktcmvr 361vplldskact dqedairkrm akfettfeel isevrtrapd arilvvgypr ifpeeptgay 421ytltasnqrw lnetiqefnq qlaeavavhd eeiaasggvg svefvdvyha ldgheigsde 481pwvngvqlrd latgvtvdrs tfhpnaaghr avgervieqi etgpgrplya tfavvagatv 541dtlagevg SEQ ID No. 6 1ggtggtgaac cagaacaccc ggtcgtcggc gtgggcgtcc aggtgcaggt gcaggttctt 61caactgctcc agcaggatgc cgccgtggcc gtgcacgatg gccttgggca ggcctgtggt 121ccccgacgag tacagcaccc atagcggatg gtcgaacggc agcggggtga actccagttc 181cgcgccttcg cccgcggctt cgaactccgc ccaggacagg gtgtcggcga cagggccgca 241gcccaggtac ggcaggacga cggtgtgctg caggctgggc atgccgtcgc gcagggcttt 301gagcacgtca cggcggtcga agtccttacc gccgtagcgg tagccgtcca cggccagcag 361cactttcggt tcgatctgcg cgaaccggtc gaggacgctg cgcaccccga agtcggggga 421acaggacgac caggtcgcac cgatcgcggc gcaggcgagg aatgcggccg tcgcctcggc 481gatgttcggc aggtaggcca cgacccggtc gccggggccc accccgaggc tgcggagggc 541cgcagcgatc gcggcggtgc gggtccgcag ttctccccag gtccactcgg tcaacggccg 601gagttcggac gcgtgccgga tcgccacggc tgatgggtca cggtcgcgga agatgtgctc 661ggcgtagttg agggtggcgc cggggaacca gacggcgccg ggcatggcgt cggaggcgag 721cactgtggtg tacggggtgg cggcgcgcac ccggtagtac tcccagatcg cggaccagaa 781tccttcgagg tcggttaccg accagcgcca cagtgcctcg tagtccggtg cgtccacacc 841gcggtgctcc cgcacccagc gggtgaacgc ggtgaggttg gcgcgttctt tgcgctcctc 901gtcgggactc cacaggatcg gcggctgcgg cttgagtgtc atgaaacgcg accccttcgt 961ggacggtgcg gatgcggtga gcgtcgggtg cctcccctaa cgctccccgg tgacggagtg 1021ttgtgcacca catctagcac gcgggacgcg gaaaccgtat ggagaaaaca cctacaaccc 1081cggccggacg gtgggtttcg gccacactta ggggtcgggt gcctgcttgc cgggcagggc 1141agtcccgggg tgctgtggtg cgggcgggag ggctgtcgct tcgaggtgtg ccggcgggac 1201actccgggcc tcagccgtac ccgcaacggg gacagttctc ctcccttccg ggctggatgg 1261tcccttcccc cgaaatgcgg cgagatctcc cagtcagccc ggaaaacacc cgctgtgccc 1321aggtactctt tgcttcgaac agacaggccg gacggtccac gggggaggtt tgtgggcagc 1381ggaccacgtg cggcgaccag acgacggttg ttcctcggta tccccgctct tgtacttgtg 1441acagcgctca cgctggtctt ggctgtcccg acggggcgcg agacgctgtg gcgcatgtgg 1501tgtgaggcca cccaggactg gtgcctgggg gtgccggtcg actcccgcgg acagcctgcg 1561gaggacggcg agtttctgct gctttctccg gtccaggcag cgacctgggg gaactattac 1621gcgctcgggg attcgtactc ttcgggggac ggggcccgcg actactatcc cggcaccgcg 1681gtgaagggcg gttgctggcg gtccgctaac gcctatccgg agctggtcgc cgaagcctac 1741gacttcgccg gacacttgtc gttcctggcc tgcagcggcc agcgcggcta cgccatgctt 1801gacgctatcg acgaggtcgg ctcgcagctg gactggaact cccctcacac gtcgctggtg 1861acgatcggga tcggcggcaa cgatctgggg ttctccacgg ttttgaagac ctgcatggtg 1921cgggtgccgc tgctggacag caaggcgtgc acggaccagg aggacgctat ccgcaagcgg 1981atggcgaaat tcgagacgac gtttgaagag ctcatcagcg aagtgcgcac ccgcgcgccg 2041gacgcccgga tccttgtcgt gggctacccc cggatttttc cggaggaacc gaccggcgcc 2101tactacacgc tgaccgcgag caaccagcgg tggctcaacg aaaccattca ggagttcaac 2161cagcagctcg ccgaggctgt cgcggtccac gacgaggaga ttgccgcgtc gggcggggtg 2221ggcagcgtgg agttcgtgga cgtctaccac gcgttggacg gccacgagat cggctcggac 2281gagccgtggg tgaacggggt gcagttgcgg gacctcgcca ccggggtgac tgtggaccgc 2341agtaccttcc accccaacgc cgctgggcac cgggcggtcg gtgagcgggt catcgagcag 2401atcgaaaccg gcccgggccg tccgctctat gccactttcg cggtggtggc gggggcgacc 2461gtggacactc tcgcgggcga ggtggggtga cccggcttac cgtccggccc gcaggtctgc 2521gagcactgcg gcgatctggt ccactgccca gtgcagttcg tcttcggtga tgaccagcgg 2581cggggagagc cggatcgttg agccgtgcgt gtctttgacg agcacacccc gctgcaggag 2641ccgttcgcac agttctcttc cggtggccag agtcgggtcg acgtcgatcc cagcccacag 2701gccgatgctg cgggccgcga ccacgccgtt gccgaccagt tggtcgaggc gggcgcgcag 2761cacgggggcg agggcgcgga catggtccag gtaagggccg tcgcggacga ggctcaccac 2821ggcagtgccg accgcgcagg cgagggcgtt gccgccgaag gtgctgccgt gctggccggg 2881gcggatcacg tcgaagactt ccgcgtcgcc taccgccgcc gccacgggca ggatgccgcc 2941gcccagcgct ttgccgaaca ggtagatatc ggcgtcgact ccgctgtggt cgcaggcccg //Thermobifida\fusca\-GDSx (SEQ ID NO: 31)

SEQ ID No. 7 1vgsgpraatr rrlflgipal vlvtaltlvl avptgretlw rmwceatqdw clgvpvdsrg 61qpaedgefll lspvqaatwg nyyalgdsys sgdgardyyp gtavkggcwr sanaypelva 121eaydfaghls flacsgqrgy amldaidevg sqldwnspht slvtigiggn dlgfstvlkt 181cmvrvpllds kactdqedai rkrmakfett feelisevrt rapdarilvv gyprifpeep 241tgayytltas nqrwlnetiq efnqqlaeav avhdeeiaas ggvgsvefvd vyhaldghei 301gsdepwvngv qlrdlatgvt vdrstfhpna aghravgerv ieqietgpgr plyatfavva 361gatvdtlage vgCorynebacterium\effciens\ GDSx (SEQ ID NO: 31) 300 aa

SEQ ID No. 8 1mrttviaasa llllagcadg areetagapp gessggiree gaeastsitd vyialgdsya 61amggrdqplr gepfclrssg nypellhaev tdltcqgavt gdlleprtlg ertlpaqvda 121ltedttlvtl siggndlgfg evagcireri agenaddcvd llgetigeql dqlppqldrv 181heairdragd aqvvvtgylp lvsagdcpel gdvseadrrw aveltgqine tvreaaerhd 241alfvlpddad ehtscappqq rwadiqgqqt dayplhptsa gheamaaavr dalglepvqp //SEQ ID No. 9 1ttctggggtg ttatggggtt gttatcggct cgtcctgggt ggatcccgcc aggtggggta 61ttcacggggg acttttgtgt ccaacagccg agaatgagtg ccctgagcgg tgggaatgag 121gtgggcgggg ctgtgtcgcc atgagggggc ggcgggctct gtggtgcccc gcgacccccg 181gccccggtga gcggtgaatg aaatccggct gtaatcagca tcccgtgccc accccgtcgg 241ggaggtcagc gcccggagtg tctacgcagt cggatcctct cggactcggc catgctgtcg 301gcagcatcgc gctcccgggt cttggcgtcc ctcggctgtt ctgcctgctg tccctggaag 361gcgaaatgat caccggggag tgatacaccg gtggtctcat cccggatgcc cacttcggcg 421ccatccggca attcgggcag ctccgggtgg aagtaggtgg catccgatgc gtcggtgacg 481ccatagtggg cgaagatctc atcctgctcg agggtgctca ggccactctc cggatcgata 541tcgggggcgt ccttgatggc gtccttgctg aaaccgaggt gcagcttgtg ggcttccaat 601ttcgcaccac ggagcgggac gaggctggaa tgacggccga agagcccgtg gtggacctca 661acgaaggtgg gtagtcccgt gtcatcattg aggaacacgc cctccaccgc acccagcttg 721tggccggagt tgtcgtaggc gctggcatcc agaagggaaa cgatctcata tttgtcggtg 781tgctcagaca tgatcttcct ttgctgtcgg tgtctggtac taccacggta gggctgaatg 841caactgttat ttttctgtta ttttaggaat tggtccatat cccacaggct ggctgtggtc 901aaatcgtcat caagtaatcc ctgtcacaca aaatgggtgg tgggagccct ggtcgcggtt 961ccgtgggagg cgccgtgccc cgcaggatcg tcggcatcgg cggatctggc cggtaccccg 1021cggtgaataa aatcattctg taaccttcat cacggttggt tttaggtatc cgcccctttc 1081gtcctgaccc cgtccccggc gcgcgggagc ccgcgggttg cggtagacag gggagacgtg 1141gacaccatga ggacaacggt catcgcagca agcgcattac tccttctcgc cggatgcgcg 1201gatggggccc gggaggagac cgccggtgca ccgccgggtg agtcctccgg gggcatccgg 1261gaggaggggg cggaggcgtc gacaagcatc accgacgtct acatcgccct cggggattcc 1321tatgcggcga tgggcgggcg ggatcagccg ttacggggtg agccgttctg cctgcgctcg 1381tccggtaatt acccggaact cctccacgca gaggtcaccg atctcacctg ccagggggcg 1441gtgaccgggg atctgctcga acccaggacg ctgggggagc gcacgctgcc ggcgcaggtg 1501gatgcgctga cggaggacac caccctggtc accctctcca tcgggggcaa tgacctcgga 1561ttcggggagg tggcgggatg catccgggaa cggatcgccg gggagaacgc tgatgattgc 1621gtggacctgc tgggggaaac catcggggag cagctcgatc agcttccccc gcagctggac 1681cgcgtgcacg aggctatccg ggaccgcgcc ggggacgcgc aggttgtggt caccggttac 1741ctgccgctcg tgtctgccgg ggactgcccc gaactggggg atgtctccga ggcggatcgt 1801cgttgggcgg ttgagctgac cgggcagatc aacgagaccg tgcgcgaggc ggccgaacga 1861cacgatgccc tctttgtcct gcccgacgat gccgatgagc acaccagttg tgcaccccca 1921cagcagcgct gggcggatat ccagggccaa cagaccgatg cctatccgct gcacccgacc 1981tccgccggcc atgaggcgat ggccgccgcc gtccgggacg cgctgggcct ggaaccggtc 2041cagccgtagc gccgggcgcg cgcttgtcga cgaccaaccc atgccaggct gcagtcacat 2101ccgcacatag cgcgcgcggg cgatggagta cgcaccatag aggatgagcc cgatgccgac 2161gatgatgagc agcacactgc cgaagggttg ttccccgagg gtgcgcagag ccgagtccag 2221acctgcggcc tgctccggat catgggccca accggcgatg acgatcaaca cccccaggat 2281cccgaaggcg ataccacggg cgacataacc ggctgttccg gtgatgatga tcgcggtccc 2341gacctgccct gaccccgcac ccgcctccag atcctcccgg aaatcccggg tggccccctt 2401ccagaggttg tagacacccg cccccagtac caccagcccg gcgaccacaa ccagcaccac 2461accccagggt tgggatagga cggtggcggt gacatcggtg gcggtctccc catcggaggt 2521gctgccgccc cgggcgaagg tggaggtggt caccgccagg gagaagtaga ccatggccat 2581gaccgccccc ttggcccttt ccttgaggtc ctcgcccgcc agcagctggc tcaattgcca 2641gagtcccagg gccgccaggg cgatgacggc aacccacagg aggaactgcc cacccggagc 2701ctccgcgatg gtggccaggg cacctgaatt cgaggcctca tcacccgaac cgccggatcc 2761agtggcgatg cgcaccgcga tccacccgat gaggatgtgc agtatgccca ggacaatgaa 2821accacctctg gccagggtgg tcagcgcggg gtggtcctcg gcctggtcgg cagcccgttc 2881gatcgtccgt ttcgcggatc tggtgtcgcc cttatccata gctcccattg aaccgccttg 2941aggggtgggc ggccactgtc agggcggatt gtgatctgaa ctgtgatgtt ccatcaaccc //S. coelicolor\ GDSx (SEQ ID NO: 31) 268 aa

NP_625998. SEQ ID No. 12 1mrrfrlvgfl sslvlaagaa ltgaataqaa qpaaadgyva lgdsyssgvg agsyisssgd 61ckrstkahpy lwaaahspst fdftacsgar tgdvlsgqlg plssgtglvs isiggndagf 121adtmttcvlq sessclsria taeayvdstl pgkldgvysa isdkapnahv vvigyprfyk 181lgttciglse tkrtainkas dhlntvlaqr aaahgftfgd vrttftghel csgspwlhsv 241nwlnigesyh ptaagqsggy lpvlngaa // SEQ ID No. 13 1cccggcggcc cgtgcaggag cagcagccgg cccgcgatgt cctcgggcgt cgtcttcatc 61aggccgtcca tcgcgtcggc gaccggcgcc gtgtagttgg cccggacctc gtcccaggtg 121cccgcggcga tctggcgggt ggtgcggtgc gggccgcgcc gaggggagac gtaccagaag 181cccatcgtca cgttctccgg ctgcggttcg ggctcgtccg ccgctccgtc cgtcgcctcg 241ccgagcacct tctcggcgag gtcggcgctg gtcgccgtca ccgtgacgtc ggcgccccgg 301ctccagcgcg agatcagcag cgtccagccg tcgccctccg ccagcgtcgc gctgcggtcg 361tcgtcgcggg cgatccgcag cacgcgcgcg ccgggcggca gcagcgtggc gccggaccgt 421acgcggtcga tgttcgccgc gtgcgagtac ggctgctcac ccgtggcgaa acggccgagg 481aacagcgcgt cgacgacgtc ggacggggag tcgctgtcgt ccacgttgag ccggatcggc 541agggcttcgt gcgggttcac ggacatgtcg ccatgatcgg gcacccggcc gccgcgtgca 601cccgctttcc cgggcacgca cgacaggggc tttctcgccg tcttccgtcc gaacttgaac 661gagtgtcagc catttcttgg catggacact tccagtcaac gcgcgtagct gctaccacgg 721ttgtggcagc aatcctgcta agggaggttc catgagacgt ttccgacttg tcggcttcct 781gagttcgctc gtcctcgccg ccggcgccgc cctcaccggg gcagcgaccg cccaggcggc 841ccaacccgcc gccgccgacg gctatgtggc cctcggcgac tcctactcct ccggggtcgg 901agcgggcagc tacatcagct cgagcggcga ctgcaagcgc agcacgaagg cccatcccta 961cctgtgggcg gccgcccact cgccctccac gttcgacttc accgcctgtt ccggcgcccg 1021tacgggtgat gttctctccg gacagctcgg cccgctcagc tccggcaccg gcctcgtctc 1081gatcagcatc ggcggcaacg acgccggttt cgccgacacc atgacgacct gtgtgctcca 1141gtccgagagc tcctgcctgt cgcggatcgc caccgccgag gcgtacgtcg actcgacgct 1201gcccggcaag ctcgacggcg tctactcggc aatcagcgac aaggcgccga acgcccacgt 1261cgtcgtcatc ggctacccgc gcttctacaa gctcggcacc acctgcatcg gcctgtccga 1321gaccaagcgg acggcgatca acaaggcctc cgaccacctc aacaccgtcc tcgcccagcg 1381cgccgccgcc cacggcttca ccttcggcga cgtacgcacc accttcaccg gccacgagct 1441gtgctccggc agcccctggc tgcacagcgt caactggctg aacatcggcg agtcgtacca 1501ccccaccgcg gccggccagt ccggtggcta cctgccggtc ctcaacggcg ccgcctgacc 1561tcaggcggaa ggagaagaag aaggagcgga gggagacgag gagtgggagg ccccgcccga 1621cggggtcccc gtccccgtct ccgtctccgt cccggtcccg caagtcaccg agaacgccac 1681cgcgtcggac gtggcccgca ccggactccg cacctccacg cgcacggcac tctcgaacgc 1741gccggtgtcg tcgtgcgtcg tcaccaccac gccgtcctgg cgcgagcgct cgccgcccga 1801cgggaaggac agcgtccgcc accccggatc ggagaccgac ccgtccgcgg tcacccaccg 1861gtagccgacc tccgcgggca gccgcccgac cgtgaacgtc gccgtgaacg cgggtgcccg 1921gtcgtgcggc ggcggacagg cccccgagta gtgggtgcgc gagcccacca cggtcacctc 1981caccgactgc gctgcggggc //S. avermitilis\ GDSx (SEQ ID NO: 31) 269 aa

NP_827753. SEQ ID No. 14 1mrrsritayv tslllavgca ltgaataqas paaaatgyva lgdsyssgvg agsylsssgd 61ckrsskaypy lwqaahspss fsfmacsgar tgdvlanqlg tlnsstglvs ltiggndagf 121sdvmttcvlq sdsaclsrin takayvdstl pgqldsvyta istkapsahv avlgyprfyk 181lggsclagls etkrsainda adylnsaiak raadhgftfg dvkstftghe icssstwlhs 241ldllnigqsy hptaagqsgg ylpvmnsva // SEQ ID No. 15 1ccaccgccgg gtcggcggcg agtctcctgg cctcggtcgc ggagaggttg gccgtgtagc 61cgttcagcgc ggcgccgaac gtcttcttca ccgtgccgcc gtactcgttg atcaggccct 121tgcccttgct cgacgcggcc ttgaagccgg tgcccttctt gagcgtgacg atgtagctgc 181ccttgatcgc ggtgggggag ccggcggcga gcaccgtgcc ctcggccggg gtggcctggg 241cgggcagtgc ggtgaatccg cccacgaggg cgccggtcgc cacggcggtt atcgcggcga 301tccggatctt cttgctacgc agctgtgcca tacgagggag tcctcctctg ggcagcggcg 361cgcctgggtg gggcgcacgg ctgtgggggg tgcgcgcgtc atcacgcaca cggccctgga 421gcgtcgtgtt ccgccctggg ttgagtaaag cctcggccat ctacgggggt ggctcaaggg 481agttgagacc ctgtcatgag tctgacatga gcacgcaatc aacggggccg tgagcacccc 541ggggcgaccc cggaaagtgc cgagaagtct tggcatggac acttcctgtc aacacgcgta 601gctggtacga cggttacggc agagatcctg ctaaagggag gttccatgag acgttcccga 661attacggcat acgtgacctc actcctcctc gccgtcggct gcgccctcac cggggcagcg 721acggcgcagg cgtccccagc cgccgcggcc acgggctatg tggccctcgg cgactcgtac 781tcgtccggtg tcggcgccgg cagctacctc agctccagcg gcgactgcaa gcgcagttcg 841aaggcctatc cgtacctctg gcaggccgcg cattcaccct cgtcgttcag tttcatggct 901tgctcgggcg ctcgtacggg tgatgtcctg gccaatcagc tcggcaccct gaactcgtcc 961accggcctgg tctccctcac catcggaggc aacgacgcgg gcttctccga cgtcatgacg 1021acctgtgtgc tccagtccga cagcgcctgc ctctcccgca tcaacacggc gaaggcgtac 1081gtcgactcca ccctgcccgg ccaactcgac agcgtgtaca cggcgatcag cacgaaggcc 1141ccgtcggccc atgtggccgt gctgggctac ccccgcttct acaaactggg cggctcctgc 1201ctcgcgggcc tctcggagac caagcggtcc gccatcaacg acgcggccga ctatctgaac 1261agcgccatcg ccaagcgcgc cgccgaccac ggcttcacct tcggcgacgt caagagcacc 1321ttcaccggcc atgagatctg ctccagcagc acctggctgc acagtctcga cctgctgaac 1381atcggccagt cctaccaccc gaccgcggcc ggccagtccg gcggctatct gccggtcatg 1441aacagcgtgg cctgagctcc cacggcctga atttttaagg cctgaatttt taaggcgaag 1501gtgaaccgga agcggaggcc ccgtccgtcg gggtctccgt cgcacaggtc accgagaacg 1561gcacggagtt ggacgtcgtg cgcaccgggt cgcgcacctc gacggcgatc tcgttcgaga 1621tcgttccgct cgtgtcgtac gtggtgacga acacctgctt ctgctgggtc tttccgccgc 1681tcgccgggaa ggacagcgtc ttccagcccg gatccgggac ctcgcccttc ttggtcaccc 1741agcggtactc cacctcgacc ggcacccggc ccaccgtgaa ggtcgccgtg aacgtgggcg 1801cctgggcggt gggcggcggg caggcaccgg agtagtcggt gtgcacgccg gtgaccgtca 1861ccttcacgga ctgggccggc ggggtcgtcg taccgccgcc gccaccgccg cctcccggag 1921tggagcccga gctgtggtcg cccccgccgt cggcgttgtc gtcctcgggg gttttcgaac //Thermobifida\fusca\-GDSx (SEQ ID NO: 31)

SEQ ID No. 16 1mgsgpraatr rrlflgipal vlvtaltlvl avptgretlw rmwceatqdw clgvpvdsrg 61qpaedgefll lspvqaatwg nyyalgdsys sgdgardyyp gtavkggcwr sanaypelva 121eaydfaghls flacsgqrgy amldaidevg sqldwnspht slvtigiggn dlgfstvlkt 181cmvrvpllds kactdqedai rkrmakfett feelisevrt rapdarilvv gyprifpeep 241tgayytltas nqrwlnetiq efnqqlaeav avhdeeiaas ggvgsvefvd vyhaldghei 301gsdepwvngv qlrdlatgvt vdrstfhpna aghravgery ieqietgpgr plyatfavva 361gatvdtlage vg //Thermobifida\fusca\-GDSx (SEQ ID NO: 31)

SEQ ID No. 17 1ctgcagacac ccgccccgcc ttctcccgga tcgtcatgtt cggcgactcc ctcagcgaca 61ccggcaagat gtactccaag atgcgcggct acctgccgtc ctccccgccg tactacgagg 121gccgcttctc gaacggcccg gtctggctgg agcagctgac gaagcagttc cccggcctga 181cgatcgccaa cgaggccgag gggggcgcga ccgcagtcgc ctacaacaag atctcctgga 241acccgaagta ccaggtcatt aacaacctcg actacgaggt cacccagttc ttgcagaagg 301actcgttcaa gcccgacgac ctggtcatcc tgtgggtggg cgccaacgac tacctggcct 361acggttggaa cacggagcag gacgccaagc gggtgcgcga cgccatctcg gacgcggcaa 421accgcatggt cctgaacggc gcgaagcaga tcctgctgtt caacctgccc gacctgggcc 481agaacccgtc cgcccgctcc cagaaggtcg tcgaggccgt ctcgcacgtg tccgcctacc 541acaacaagct gctcctcaac ctcgcccggc agctcgcccc gacgggcatg gtcaagctgt 601tcgagatcga caagcagttc gcggagatgc tgcgcgaccc ccagaacttc ggcctgagcg 661acgtggagaa cccgtgctac gacggcggct acgtgtggaa gccgttcgcc acccggtccg 721tctcgaccga ccggcagctg tcggccttct cgccccagga gcgcctggcg atcgctggca 781acccgctcct ggcacaggcg gtagcttcgc cgatggcccg ccgctcggcc tcgcccctca 841actgcgaggg caagatgttc tgggaccagg tccaccccac caccgtggtc cacgccgccc 901tctcggagcg cgccgccacc ttcatcgaga cccagtacga gttcctcgcc cactagtcta 961gaggatcc

Thus, in a further aspect, the present invention provides the use of alipolytic enzyme comprising any one of the amino acid sequences shown asSEQ ID No. 4, 5, 7, 8, 12, 14, or 16 or an amino acid sequence which hasat least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identitytherewith, or encoded by any one of the nucleotide sequences shown asSEQ ID No. 3, 6, 9, 13, 15 or 17 or a nucleotide sequence which has atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identity therewith,in a foodstuff for the preparation of a lyso-glycolipid, for exampledigalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG)by treatment of a glycolipid (e.g. digalactosyl diglyceride (DGDG) ormonogalactosyl diglyceride (MGDG)) with the lipolytic enzyme accordingto the present invention to produce the partial hydrolysis product, i.e.the lyso-glycolipid.

In a further aspect, the present invention yet further provides the useof a lipolytic enzyme comprising any one of the amino acid sequencesshown as SEQ ID No. 4, 5, 7, 8, 12, 14 or 16 or an amino acid sequencewhich has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98%identity therewith, or encoded by any one of the nucleotide sequencesshown as SEQ ID No. 3, 6, 9, 13, 15 or 17 or a nucleotide sequence whichhas at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identitytherewith, in a foodstuff for the preparation of a lyso-phospholipid,for example lysolecithin, by treatment of a phospholipid (e.g. lecithin)with the enzyme to produce the partial hydrolysis product, i.e. alyso-phospholipid.

In another aspect, the present invention yet further provides the use ofa lipolytic enzyme comprising any one of the amino acid sequences shownas SEQ ID No. 4, 5, 7, 8, 12, 14 or 16 or an amino acid sequence whichhas at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identitytherewith, or encoded by any one of the nucleotide sequences shown asSEQ ID No. 3, 6, 9, 13, 15 or 17 or a nucleotide sequence which has atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identity therewith,in an egg or an egg-based product for the hydrolysis of phospholipidsand/or glycolipids.

In another aspect the present invention provides the use of a lipolyticenzyme comprising any one of the amino acid sequences shown as SEQ IDNo. 4, 5, 7, 8, 12, 14 or 16 or an amino acid sequence which has atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identity therewith,or encoded by any one of the nucleotide sequences shown as SEQ ID No. 3,6, 9, 13, 15 or 17 or a nucleotide sequence which has at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97% or 98% identity therewith, in a substrate(preferably a foodstuff) for hydrolysing fatty acyl groups.

In another aspect the present invention provides the use of a lipolyticenzyme comprising any one of the amino acid sequences shown as SEQ IDNo. 4, 5, 7, 8, 12, 14 or 16 or an amino acid sequence which has atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identity therewith,or encoded by any one of the nucleotide sequences shown as SEQ ID No. 3,6, 9, 13, 15 or 17 or a nucleotide sequence which has at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97% or 98% identity therewith, in an edible oilfor reducing the content of a phospholipid.

In a further aspect the present invention relates to the use of thelipolytic enzyme comprising any one of the amino acid sequences shown asSEQ ID No. 4, 5, 7, 8, 12, 14 or 16 or an amino acid sequence which hasat least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identitytherewith, or encoded by any one of the nucleotide sequences shown asSEQ ID No. 3, 6, 9, 13, 15 or 17 or a nucleotide sequence which has atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% identity therewith,in a substrate (preferably a bioconversion mixture comprising polarlipids (preferably glycolipids)) for the production of make high valueproducts, such as carbohydrate esters and/or protein esters and/orprotein subunit esters and/or a hydroxy acid ester.

In a preferable aspect, the present invention relates to a lipolyticenzyme comprising any one of amino sequences shown as SEQ ID No. 8, 14or 16 or an amino acid sequence which has at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97% or 98% identity therewith for the uses describedherein.

More preferably the present invention relates to the use of a lipolyticenzyme comprising the amino acid sequence shown as SEQ ID No. 16 or anamino acid sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97% or 98% identity therewith.

In a broad aspect the present invention may provide a lipolytic enzymecapable of hydrolysing at least a glycolipid and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptors, wherein the enzyme is obtainable, preferably obtained,from Thermobifida spp, preferably T. fusca.

In another broad aspect the present invention may provide a lipolyticenzyme capable of hydrolysing at least a glycolipid and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptors, wherein the enzyme is obtainable, preferably obtained,from Corynebacterium spp, preferably C. efficiens.

In another broad aspect the present invention may provide a lipolyticenzyme capable of hydrolysing at least a glycolipid and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptors, wherein the enzyme is obtainable, preferably obtained,from Streptomyces avermitilis.

In a further aspect the present invention may provide a lipolytic enzymecapable of hydrolysing at least a glycolipid and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptors, wherein the enzyme comprises SEQ ID No. 5, 7, 8, 12, or16 or an amino acid sequence which has at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97% or 98% identity therewith, or the enzyme is encoded by anyone of the nucleotide sequences shown as SEQ ID No. 6, 9, 13, or 17 or anucleotide sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97% or 98% identity therewith.

In a further aspect the present invention may provide a lipolytic enzymecapable of hydrolysing at least a glycolipid and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptors, wherein the enzyme comprises SEQ ID No. 14 or an aminoacid sequence which has at least 80%, 85%, 90%, 95%, 96%, 97% or 98%identity therewith, or the enzyme is encoded by any one of thenucleotide sequences shown as SEQ ID No. 15 or a nucleotide sequencewhich has at least 80%, 85%, 90%, 95%, 96%, 97% or 98% identitytherewith.

In a further aspect the present invention may provide a lipolytic enzymecapable of hydrolysing at least a glycolipid and/or capable oftransferring an acyl group from at least a galactolipid to one or moreacyl acceptors, wherein the enzyme comprises SEQ ID No. 16 or an aminoacid sequence which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%or 98% identity therewith, or the enzyme is encoded by any one of thenucleotide sequences shown as SEQ ID No. 17 or a nucleotide sequencewhich has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98%identity therewith.

In one embodiment of the present invention preferably the Streptomycesspecies from which the lipolytic enzyme is obtainable (or obtained) isnot Streptomyces rimosus.

In one embodiment of the present invention preferably the Streptomycesspecies from which the lipolytic enzyme is obtainable (or obtained) isnot Streptomyces coelicolor.

ADVANTAGES

One advantage of the present invention is that the lipolytic enzyme hassignificant glycolipid hydrolysing activity. This was surprising for alipolytic enzyme from Streptomyces spp. In addition, this was surprisingfor a lipolytic enzyme from Thermobifida and Corynebacterium spp.

A further advantage of the present invention is that the lipolyticenzyme has no or no significant triacylglycerol hydrolysing activity.

Isolated

In one aspect, preferably the sequence is in an isolated form. The term“isolated” means that the sequence is at least substantially free fromat least one other component with which the sequence is naturallyassociated in nature and as found in nature.

Purified

In one aspect, preferably the sequence is in a purified form. The term“purified” means that the sequence is in a relatively pure state—e.g. atleast about 90% pure, or at least about 95% pure or at least about 98%pure.

Nucleotide Sequence

The scope of the present invention encompasses nucleotide sequencesencoding enzymes having the specific properties as defined herein.

The term “nucleotide sequence” as used herein refers to anoligonucleotide sequence or polynucleotide sequence, and variants,homologues, fragments and derivatives thereof (such as portionsthereof). The nucleotide sequence may be of genomic or synthetic orrecombinant origin, which may be double-stranded or single-strandedwhether representing the sense or anti-sense strand.

The term “nucleotide sequence” in relation to the present inventionincludes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it meansDNA, more preferably cDNA sequence coding for the present invention.

In a preferred embodiment, the nucleotide sequence when relating to andwhen encompassed by the per se scope of the present invention does notinclude the native nucleotide sequence according to the presentinvention when in its natural environment and when it is linked to itsnaturally associated sequence(s) that is/are also in its/their naturalenvironment. For ease of reference, we shall call this preferredembodiment the “non-native nucleotide sequence”. In this regard, theterm “native nucleotide sequence” means an entire nucleotide sequencethat is in its native environment and when operatively linked to anentire promoter with which it is naturally associated, which promoter isalso in its native environment. However, the amino acid sequenceencompassed by scope the present invention can be isolated and/orpurified post expression of a nucleotide sequence in its nativeorganism. Preferably, however, the amino acid sequence encompassed byscope of the present invention may be expressed by a nucleotide sequencein its native organism but wherein the nucleotide sequence is not underthe control of the promoter with which it is naturally associated withinthat organism.

Preparation of the Nucleotide Sequence

Typically, the nucleotide sequence encompassed by scope of the presentinvention is prepared using recombinant DNA techniques (i.e. recombinantDNA). However, in an alternative embodiment of the invention, thenucleotide sequence could be synthesised, in whole or in part, usingchemical methods well known in the art (see Caruthers M H et al., (1980)Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids ResSymp Ser 225-232).

A nucleotide sequence encoding an enzyme which has the specificproperties as defined herein may be identified and/or isolated and/orpurified from any cell or organism producing said enzyme. Variousmethods are well known within the art for the identification and/orisolation and/or purification of nucleotide sequences. By way ofexample, PCR amplification techniques to prepare more of a sequence maybe used once a suitable sequence has been identified and/or isolatedand/or purified.

By way of further example, a genomic DNA and/or cDNA library may beconstructed using chromosomal DNA or messenger RNA from the organismproducing the enzyme. If the amino acid sequence of the enzyme or a partof the amino acid sequence of the enzyme is known, labelledoligonucleotide probes may be synthesised and used to identifyenzyme-encoding clones from the genomic library prepared from theorganism. Alternatively, a labelled oligonucleotide probe containingsequences homologous to another known enzyme gene could be used toidentify enzyme-encoding clones. In the latter case, hybridisation andwashing conditions of lower stringency are used.

Alternatively, enzyme-encoding clones could be identified by insertingfragments of genomic DNA into an expression vector, such as a plasmid,transforming enzyme-negative bacteria with the resulting genomic DNAlibrary, and then plating the transformed bacteria onto agar platescontaining a substrate for the enzyme (e.g. maltose for a glucosidase(maltase) producing enzyme), thereby allowing clones expressing theenzyme to be identified.

In a yet further alternative, the nucleotide sequence encoding theenzyme may be prepared synthetically by established standard methods,e.g. the phosphoroamidite method described by Beucage S. L. et al.,(1981) Tetrahedron Letters 22, p 1859-1869, or the method described byMatthes et al., (1984) EMBO J. 3, p 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesised, e.g. in an automatic DNAsynthesiser, purified, annealed, ligated and cloned in appropriatevectors.

The nucleotide sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin, or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate) in accordance with standard techniques. Each ligatedfragment corresponds to various parts of the entire nucleotide sequence.The DNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).

Due to degeneracy in the genetic code, nucleotide sequences may bereadily produced in which the triplet codon usage, for some or all ofthe amino acids encoded by the original nucleotide sequence, has beenchanged thereby producing a nucleotide sequence with low homology to theoriginal nucleotide sequence but which encodes the same, or a variant,amino acid sequence as encoded by the original nucleotide sequence. Forexample, for most amino acids the degeneracy of the genetic code is atthe third position in the triplet codon (wobble position) (for referencesee Stryer, Lubert, Biochemistry, Third Edition, Freeman Press, ISBN0-7167-1920-7) therefore, a nucleotide sequence in which all tripletcodons have been “wobbled” in the third position would be about 66%identical to the original nucleotide sequence however, the amendednucleotide sequence would encode for the same, or a variant, primaryamino acid sequence as the original nucleotide sequence.

Therefore, the present invention further relates to any nucleotidesequence that has alternative triplet codon usage for at least one aminoacid encoding triplet codon, but which encodes the same, or a variant,polypeptide sequence as the polypeptide sequence encoded by the originalnucleotide sequence.

Furthermore, specific organisms typically have a bias as to whichtriplet codons are used to encode amino acids. Preferred codon usagetables are widely available, and can be used to prepare codon optimisedgenes. Such codon optimisation techniques are routinely used to optimiseexpression of transgenes in a heterologous host.

Molecular Evolution

Once an enzyme-encoding nucleotide sequence has been isolated, or aputative enzyme-encoding nucleotide sequence has been identified, it maybe desirable to modify the selected nucleotide sequence, for example itmay be desirable to mutate the sequence in order to prepare an enzyme inaccordance with the present invention.

Mutations may be introduced using synthetic oligonucleotides. Theseoligonucleotides contain nucleotide sequences flanking the desiredmutation sites.

A suitable method is disclosed in Morinaga et al (Biotechnology (1984)2, p 646-649). Another method of introducing mutations intoenzyme-encoding nucleotide sequences is described in Nelson and Long(Analytical Biochemistry (1989), 180, p 147-151).

Instead of site directed mutagenesis, such as described above, one canintroduce mutations randomly for instance using a commercial kit such asthe GeneMorph PCR mutagenesis kit from Stratagene, or the Diversify PCRrandom mutagenesis kit from Clontech. EP 0 583 265 refers to methods ofoptimising PCR based mutagenesis, which can also be combined with theuse of mutagenic DNA analogues such as those described in EP 0 866 796.Error prone PCR technologies are suitable for the production of variantsof lipolytic enzymes with preferred characteristics. WO0206457 refers tomolecular evolution of lipases.

A third method to obtain novel sequences is to fragment non-identicalnucleotide sequences, either by using any number of restriction enzymesor an enzyme such as Dnase I, and reassembling full nucleotide sequencescoding for functional proteins. Alternatively one can use one ormultiple non-identical nucleotide sequences and introduce mutationsduring the reassembly of the full nucleotide sequence. DNA shuffling andfamily shuffling technologies are suitable for the production ofvariants of lipid acyl transferases with preferred characteristics.Suitable methods for performing ‘shuffling’ can be found in EP0 752 008,EP1 138 763, EP1 103 606. Shuffling can also be combined with otherforms of DNA mutagenesis as described in U.S. Pat. No. 6,180,406 and WO01/34835.

Thus, it is possible to produce numerous site directed or randommutations into a nucleotide sequence, either in vivo or in vitro, and tosubsequently screen for improved functionality of the encodedpolypeptide by various means. Using in silico and exo mediatedrecombination methods (see WO 00/58517, U.S. Pat. No. 6,344,328, U.S.Pat. No. 6,361,974), for example, molecular evolution can be performedwhere the variant produced retains very low homology to known enzymes orproteins. Such variants thereby obtained may have significant structuralanalogy to known lipolytic enzymes, but have very low amino acidsequence homology.

As a non-limiting example, In addition, mutations or natural variants ofa polynucleotide sequence can be recombined with either the wild type orother mutations or natural variants to produce new variants. Such newvariants can also be screened for improved functionality of the encodedpolypeptide.

The application of the above-mentioned and similar molecular evolutionmethods allows the identification and selection of variants of theenzymes of the present invention which have preferred characteristicswithout any prior knowledge of protein structure or function, and allowsthe production of non-predictable but beneficial mutations or variants.There are numerous examples of the application of molecular evolution inthe art for the optimisation or alteration of enzyme activity, suchexamples include, but are not limited to one or more of the following:optimised expression and/or activity in a host cell or in vitro,increased enzymatic activity, altered substrate and/or productspecificity, increased or decreased enzymatic or structural stability,altered enzymatic activity/specificity in preferred environmentalconditions, e.g. temperature, pH, substrate

As will be apparent to a person skilled in the art, using molecularevolution tools an enzyme may be altered to improve the functionality ofthe enzyme.

Suitably, the lipolytic enzyme used in the invention may be a variant,i.e. may contain at least one amino acid substitution, deletion oraddition, when compared to a parental enzyme. Variant enzymes retain atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% homologywith the parent enzyme. Suitable parent enzymes may include any enzymewith esterase or lipase activity. Preferably, the parent enzyme alignsto the pfam00657 consensus sequence.

In a preferable embodiment a variant lipolytic enzyme retains orincorporates at least one or more of the pfam00657 consensus sequenceamino acid residues found in the GDSx (SEQ ID NO: 31), GANDY (SEQ ID NO:35) and HPT blocks.

Enzymes, such as lipases with no or low galactolipase and/orphospholipase activity in an aqueous environment may be mutated usingmolecular evolution tools to introduce or enhance the galactolipaseand/or phospholipase activity, thereby producing a lipolytic enzyme withsignificant galactolipase and/or phospholipase activity suitable for usein the compositions and methods of the present invention.

Suitably the variant enzyme may have no activity on triglycerides and/ormonoglycerides and/or diglycerides.

Alternatively, the variant enzyme for use in the invention may haveincreased activity on triglycerides, and/or may also have increasedactivity on one or more of the following, polar lipids, phospholipids,lecithin, phosphatidylcholine, glycolipids, digalactosyl monoglyceride,monogalactosyl monoglyceride.

Amino Acid Sequences

The scope of the present invention also encompasses amino acid sequencesof enzymes having the specific properties as defined herein.

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”. Insome instances, the term “amino acid sequence” is synonymous with theterm “enzyme”.

The amino acid sequence may be prepared/isolated from a suitable source,or it may be made synthetically or it may be prepared by use ofrecombinant DNA techniques.

The enzyme encompassed in the present invention may be used inconjunction with other enzymes. Thus the present invention also covers acombination of enzymes wherein the combination comprises the enzyme ofthe present invention and another enzyme, which may be another enzymeaccording to the present invention. This aspect is discussed in a latersection.

Preferably the amino acid sequence when relating to and when encompassedby the per se scope of the present invention is not a native enzyme. Inthis regard, the term “native enzyme” means an entire enzyme that is inits native environment and when it has been expressed by its nativenucleotide sequence.

Identity/Homology

The present invention also encompasses the use of homologues of anyamino acid sequence of an enzyme or of any nucleotide sequence encodingsuch an enzyme.

Here, the term “homologue” means an entity having a certain homologywith the amino acid sequences and the nucleotide sequences. Here, theterm “homology” can be equated with “identity”. These terms will be usedinterchangeably herein.

In the present context, a homologous amino acid sequence is taken toinclude an amino acid sequence which may be at least 87 or 90%identical, preferably at least 95, 96, 97, 98 or 99% identical to thesequence. Typically, the homologues will comprise the same active sitesetc.—e.g. as the subject amino acid sequence. Although homology can alsobe considered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

In the present context, an homologous nucleotide sequence is taken toinclude a nucleotide sequence which may be at least 85 or 90% identical,preferably at least 95, 96, 97, 98 or 99% identical to a nucleotidesequence encoding an enzyme of the present invention (the subjectsequence). Typically, the homologues will comprise the same sequencesthat code for the active sites etc. as the subject sequence. Althoughhomology can also be considered in terms of similarity (i.e. amino acidresidues having similar chemical properties/functions), in the contextof the present invention it is preferred to express homology in terms ofsequence identity.

For the amino acid sequences and the nucleotide sequences, homologycomparisons can be conducted by eye, or more usually, with the aid ofreadily available sequence comparison programs. These commerciallyavailable computer programs can calculate % homology between two or moresequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc.Acids Research 12 p 387). Examples of other software than can performsequence comparisons include, but are not limited to, the BLAST package(see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4^(th)Ed—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) andthe GENEWORKS suite of comparison tools. Both BLAST and FASTA areavailable for offline and online searching (see Ausubel et al., 1999,Short Protocols in Molecular Biology, pages 7-58 to 7-60).

However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999174 (2): 247-50; and FEMS Microbiol Lett 1999 177 (1): 187-8).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe case of other software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in DNASIS™ (Hitachi Software), based on analgorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene 73(1), 237-244).

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

In a preferable aspect of the present invention the following softwareand settings for calculating percentage sequence homology/identity areused. For amino acid sequences percentage of identities (homology) or“positives” are calculated by the AlignX Vector NTI (Vector NTI Advance9.1 from Invitrogen Corporation, Carlsbad, Calif., USA.) for eachpossible pair of amino acid sequences. Settings are default parameters(Gap opening penalty −10, Gap extension penalty 0.1).

For nucleic acid sequences percentage of identities (homology) or“positives” are calculated by the AlignX VectorNTI programme fromInformax Inc. (USA) for each possible pair of nucleic acid sequences.Settings are default settings which for DNA is: Gap opening penalty: 15and Gap extension penalty: 6.66. (same settings for multiplealignments).

Preferably the amino acid identity (homology) is calculated across thefull-length amino acid sequence (e.g. SEQ IDs 4, 5, 7, 8, 10, 12 and14), or for nucleic acid to a corresponding polynucleotide which encodesthe respective the full-length amino acid sequence. Amino acid ornucleic acid identity (homology) may be, preferably, calculated bycomparing the homology/identity over the mature polypeptide sequence,i.e. a polypeptide sequence which has been co- or post-translationallyprocessed, for example by cleavage of an N-terminal signal peptide, or aC-terminal cleavage event.

The sequences may also have deletions, insertions or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in amino acid properties (such aspolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues) and it is therefore useful to groupamino acids together in functional groups. Amino acids can be groupedtogether based on the properties of their side chain alone. However itis more useful to include mutation data as well. The sets of amino acidsthus derived are likely to be conserved for structural reasons. Thesesets can be described in the form of a Venn diagram (Livingstone C. D.and Barton G. J. (1993) “Protein sequence alignments: a strategy for thehierarchical analysis of residue conservation” Comput. Appl Biosci. 9:745-756) (Taylor W. R. (1986) “The classification of amino acidconservation” J. Theor. Biol. 119; 205-218). Conservative substitutionsmay be made, for example according to the table below which describes agenerally accepted Venn diagram grouping of amino acids.

SET SUB-SET Hydro- F W Y H K M I L V A G C Aromatic F W Y H phobicAliphatic I L V Polar W Y H K R E D C S T N Q Charged H K R E DPositively H K R charged Negatively E D charged Small V C A G S P T N DTiny AGS

The present invention also encompasses homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) that may occur i.e. like-for-like substitution such as basicfor basic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Replacements may also be made by unnatural amino acids.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or β-alanine residues. Afurther form of variation involves the presence of one or more aminoacid residues in peptoid form, and will be well understood by thoseskilled in the art. For the avoidance of doubt, “the peptoid form” isused to refer to variant amino acid residues wherein the α-carbonsubstituent group is on the residue's nitrogen atom rather than theα-carbon. Processes for preparing peptides in the peptoid form are knownin the art, for example Simon R J et al., PNAS (1992) 89 (20), 9367-9371and Horwell D C, Trends Biotechnol. (1995) 13 (4), 132-134.

The nucleotide sequences for use in the present invention may includewithin them synthetic or modified nucleotides. A number of differenttypes of modification to oligonucleotides are known in the art. Theseinclude methylphosphonate and phosphorothioate backbones and/or theaddition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the nucleotide sequences described herein may bemodified by any method available in the art. Such modifications may becarried out in order to enhance the in vivo activity or life span ofnucleotide sequences of the present invention.

The present invention also encompasses the use of nucleotide sequencesthat are complementary to the sequences presented herein, or anyderivative, fragment or derivative thereof. If the sequence iscomplementary to a fragment thereof then that sequence can be used as aprobe to identify similar coding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained for example by probing DNA libraries made from arange of individuals, for example individuals from differentpopulations. In addition, other homologues may be obtained and suchhomologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein. Such sequences may be obtained by probing cDNA libraries madefrom or genomic DNA libraries from other species, and probing suchlibraries with probes comprising all or part of any one of the sequencesin the attached sequence listings under conditions of medium to highstringency. Similar considerations apply to obtaining species homologuesand allelic variants of the polypeptide or nucleotide sequences of theinvention.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of characterised sequences. This may be useful where forexample silent codon sequence changes are required to optimise codonpreferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

Polynucleotides (nucleotide sequences) of the invention may be used toproduce a primer, e.g. a PCR primer, a primer for an alternativeamplification reaction, a probe e.g. labelled with a revealing label byconventional means using radioactive or non-radioactive labels, or thepolynucleotides may be cloned into vectors. Such primers, probes andother fragments will be at least 15, preferably at least 20, for exampleat least 25, 30 or 40 nucleotides in length, and are also encompassed bythe term polynucleotides of the invention as used herein.

Polynucleotides such as DNA polynucleotides and probes according to theinvention may be produced recombinantly, synthetically, or by any meansavailable to those of skill in the art. They may also be cloned bystandard techniques.

In general, primers will be produced by synthetic means, involving astepwise manufacture of the desired nucleic acid sequence one nucleotideat a time. Techniques for accomplishing this using automated techniquesare readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. The primers may be designed to contain suitable restrictionenzyme recognition sites so that the amplified DNA can be cloned into asuitable cloning vector.

Biologically Active

Preferably, the variant sequences etc. are at least as biologicallyactive as the sequences presented herein.

As used herein “biologically active” refers to a sequence having asimilar structural function (but not necessarily to the same degree),and/or similar regulatory function (but not necessarily to the samedegree), and/or similar biochemical function (but not necessarily to thesame degree) of the naturally occurring sequence.

Hybridisation

The present invention also encompasses sequences that are complementaryto the nucleic acid sequences of the present invention or sequences thatare capable of hybridising either to the sequences of the presentinvention or to sequences that are complementary thereto.

The term “hybridisation” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction (PCR) technologies.

The present invention also encompasses the use of nucleotide sequencesthat are capable of hybridising to the sequences that are complementaryto the sequences presented herein, or any derivative, fragment orderivative thereof.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hybridising to the nucleotide sequencespresented herein.

Preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hybridising understringent conditions (e.g. 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein.

More preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hybridising under highstringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein.

The present invention also relates to nucleotide sequences that canhybridise to the nucleotide sequences of the present invention(including complementary sequences of those presented herein).

The present invention also relates to nucleotide sequences that arecomplementary to sequences that can hybridise to the nucleotidesequences of the present invention (including complementary sequences ofthose presented herein).

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridising to thenucleotide sequences presented herein under conditions of intermediateto maximal stringency.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to the nucleotide sequence of the present invention,or the complement thereof, under stringent conditions (e.g. 50° C. and0.2×SSC).

In a more preferred aspect, the present invention covers nucleotidesequences that can hybridise to the nucleotide sequence of the presentinvention, or the complement thereof, under high stringent conditions(e.g. 65° C. and 0.1×SSC).

Recombinant

In one aspect the sequence for use in the present invention is arecombinant sequence—i.e. a sequence that has been prepared usingrecombinant DNA techniques.

These recombinant DNA techniques are within the capabilities of a personof ordinary skill in the art. Such techniques are explained in theliterature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis,1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,Cold Spring Harbor Laboratory Press.

Synthetic

In one aspect the sequence for use in the present invention is asynthetic sequence—i.e. a sequence that has been prepared by in vitrochemical or enzymatic synthesis. It includes, but is not limited to,sequences made with optimal codon usage for host organisms—such as themethylotrophic yeasts Pichia and Hansenula.

Expression of Enzymes

The nucleotide sequence for use in the present invention may beincorporated into a recombinant replicable vector. The vector may beused to replicate and express the nucleotide sequence, in enzyme form,in and/or from a compatible host cell.

Expression may be controlled using control sequences e.g. regulatorysequences.

The enzyme produced by a host recombinant cell by expression of thenucleotide sequence may be secreted or may be contained intracellularlydepending on the sequence and/or the vector used. The coding sequencesmay be designed with signal sequences which direct secretion of thesubstance coding sequences through a particular prokaryotic oreukaryotic cell membrane.

Expression Vector

The term “expression vector” means a construct capable of in vivo or invitro expression.

Preferably, the expression vector is incorporated into the genome of asuitable host organism. The term “incorporated” preferably covers stableincorporation into the genome.

The nucleotide sequence of the present invention may be present in avector in which the nucleotide sequence is operably linked to regulatorysequences capable of providing for the expression of the nucleotidesequence by a suitable host organism.

The vectors for use in the present invention may be transformed into asuitable host cell as described below to provide for expression of apolypeptide of the present invention.

The choice of vector e.g. a plasmid, cosmid, or phage vector will oftendepend on the host cell into which it is to be introduced.

The vectors for use in the present invention may contain one or moreselectable marker genes-such as a gene, which confers antibioticresistance e.g. ampicillin, kanamycin, chloramphenicol or tetracyclinresistance. Alternatively, the selection may be accomplished byco-transformation (as described in WO91/17243).

Vectors may be used in vitro, for example for the production of RNA orused to transfect, transform, transduce or infect a host cell.

Thus, in a further embodiment, the invention provides a method of makingnucleotide sequences of the present invention by introducing anucleotide sequence of the present invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector.

The vector may further comprise a nucleotide sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and pIJ702.

Regulatory Sequences

In some applications, the nucleotide sequence for use in the presentinvention is operably linked to a regulatory sequence which is capableof providing for the expression of the nucleotide sequence, such as bythe chosen host cell. By way of example, the present invention covers avector comprising the nucleotide sequence of the present inventionoperably linked to such a regulatory sequence, i.e. the vector is anexpression vector.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

The term “regulatory sequences” includes promoters and enhancers andother expression regulation signals.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site.

Enhanced expression of the nucleotide sequence encoding the enzyme ofthe present invention may also be achieved by the selection ofheterologous regulatory regions, e.g. promoter, secretion leader andterminator regions.

Preferably, the nucleotide sequence according to the present inventionis operably linked to at least a promoter.

Examples of suitable promoters for directing the transcription of thenucleotide sequence in a bacterial, fungal or yeast host are well knownin the art.

Constructs

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes a nucleotide sequence for use accordingto the present invention directly or indirectly attached to a promoter.

An example of an indirect attachment is the provision of a suitablespacer group such as an intron sequence, such as the Sh1-intron or theADH intron, intermediate the promoter and the nucleotide sequence of thepresent invention. The same is true for the term “fused” in relation tothe present invention which includes direct or indirect attachment. Insome cases, the terms do not cover the natural combination of thenucleotide sequence coding for the protein ordinarily associated withthe wild type gene promoter and when they are both in their naturalenvironment.

The construct may even contain or express a marker, which allows for theselection of the genetic construct.

For some applications, preferably the construct of the present inventioncomprises at least the nucleotide sequence of the present inventionoperably linked to a promoter.

Host Cells

The term “host cell”—in relation to the present invention includes anycell that comprises either the nucleotide sequence or an expressionvector as described above and which is used in the recombinantproduction of an enzyme having the specific properties as definedherein.

Thus, a further embodiment of the present invention provides host cellstransformed or transfected with a nucleotide sequence that expresses theenzyme of the present invention. The cells will be chosen to becompatible with the said vector and may for example be prokaryotic (forexample bacterial), fungal, yeast or plant cells. Preferably, the hostcells are not human cells.

Examples of suitable bacterial host organisms are gram positive or gramnegative bacterial species.

Depending on the nature of the nucleotide sequence encoding the enzymeof the present invention, and/or the desirability for further processingof the expressed protein, eukaryotic hosts such as yeasts or other fungimay be preferred. In general, yeast cells are preferred over fungalcells because they are easier to manipulate. However, some proteins areeither poorly secreted from the yeast cell, or in some cases are notprocessed properly (e.g. hyperglycosylation in yeast). In theseinstances, a different fungal host organism should be selected.

The use of suitable host cells—such as yeast, fungal and plant hostcells—may provide for post-translational modifications (e.g.myristoylation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products of the presentinvention.

The host cell may be a protease deficient or protease minus strain.

The genotype of the host cell may be modified to improve expression.

Examples of host cell modifications include protease deficiency,supplementation of rare tRNA's, and modification of the reductivepotential in the cytoplasm to enhance disulphide bond formation.

For example, the host cell E. coli may overexpress rare tRNA's toimprove expression of heterologous proteins as exemplified/described inKane (Curr Opin Biotechnol (1995), 6, 494-500 “Effects of rare codonclusters on high-level expression of heterologous proteins in E. coli”).The host cell may be deficient in a number of reducing enzymes thusfavouring formation of stable disulphide bonds as exemplified/describedin Bessette (Proc Natl Acad Sci USA (1999), 96, 13703-13708 “Efficientfolding of proteins with multiple disulphide bonds in the Escherichiacoli cytoplasm”).

In one embodiment the host cell is a bacteria, preferably agram-positive bacteria, preferably a host cell selected fromActinobacteria, such as Biofidobacteria and Aeromonas, particularlypreferably Aeromonas salmonicida. Still more preferred areActinomicetales such as Corynebacteria, in particular Corynebacteriumglutamicum and Nocardia. Particularly preferred are Streptomycetaceae,such as Streptomyces, especially S. lividans.

A microbial host can be used for expression of the galactolipase gene,e.g. Eubacteria, Archea or Fungi, including yeast. Preferred areEubacteria, for example, Firmicutes (low GC-Gram positive bacteria),such as Bacillus subtilis and other bacillus species, lactic acidbacteria such as species of genera Lactobacillus and Lactococcus.

Also preferred are Gram-negative Proteobacteria, in particularGammaproteobacteria, such as host species belonging to the generaPseudomonas, Xanthomonas, Citrobacter and Escherichia, especiallyEscherichia coli.

In another embodiment the host cell is the same genus as the native hostspecies, i.e. the recombinant gene is re-introduced and expressed in aspecies from the same genus as the species from which the recombinantgene was isolated.

In another embodiment the host cell is the native host species, i.e. therecombinant gene is re-introduced and expressed in the same species fromwhich the recombinant gene was isolated.

Organism

The term “organism” in relation to the present invention includes anyorganism that could comprise the nucleotide sequence coding for theenzyme according to the present invention and/or products obtainedtherefrom, and/or wherein a promoter can allow expression of thenucleotide sequence according to the present invention when present inthe organism.

Suitable organisms may include a prokaryote, fungus, yeast or a plant.

The term “transgenic organism” in relation to the present inventionincludes any organism that comprises the nucleotide sequence coding forthe enzyme according to the present invention and/or the productsobtained therefrom, and/or wherein a promoter can allow expression ofthe nucleotide sequence according to the present invention within theorganism. Preferably the nucleotide sequence is incorporated in thegenome of the organism.

The term “transgenic organism” does not cover native nucleotide codingsequences in their natural environment when they are under the controlof their native promoter which is also in its natural environment.

Therefore, the transgenic organism of the present invention includes anorganism comprising any one of, or combinations of, the nucleotidesequence coding for the enzyme according to the present invention,constructs according to the present invention, vectors according to thepresent invention, plasmids according to the present invention, cellsaccording to the present invention, tissues according to the presentinvention, or the products thereof.

For example the transgenic organism may also comprise the nucleotidesequence coding for the enzyme of the present invention under thecontrol of a heterologous promoter.

Transformation of Host Cells/Organism

As indicated earlier, the host organism can be a prokaryotic or aeukaryotic organism. Examples of suitable prokaryotic hosts include E.coli and Bacillus subtilis.

Teachings on the transformation of prokaryotic hosts is well documentedin the art, for example see Sambrook et al (Molecular Cloning: ALaboratory Manual, 2nd edition, 1989, Cold Spring Harbor LaboratoryPress). If a prokaryotic host is used then the nucleotide sequence mayneed to be suitably modified before transformation—such as by removal ofintrons.

Filamentous fungi cells may be transformed using various methods knownin the art—such as a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known. The use of Aspergillus as a host microorganismis described in EP 0 238 023.

Another host organism can be a plant. A review of the general techniquesused for transforming plants may be found in articles by Potrykus (AnnuRev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou(Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachingson plant transformation may be found in EP-A-0449375.

General teachings on the transformation of fungi, yeasts and plants arepresented in following sections.

Transformed Fungus

A host organism may be a fungus—such as a filamentous fungus. Examplesof suitable such hosts include any member belonging to the generaThermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora,Trichoderma and the like.

Teachings on transforming filamentous fungi are reviewed in U.S. Pat.No. 5,741,665 which states that standard techniques for transformationof filamentous fungi and culturing the fungi are well known in the art.An extensive review of techniques as applied to N. crassa is found, forexample in Davis and de Serres, Methods Enzymol (1971) 17A: 79-143.

Further teachings on transforming filamentous fungi are reviewed in U.S.Pat. No. 5,674,707.

In one aspect, the host organism can be of the genus Aspergillus, suchas Aspergillus niger.

A transgenic Aspergillus according to the present invention can also beprepared by following, for example, the teachings of Turner G. 1994(Vectors for genetic manipulation. In: Martinelli S. D., Kinghorn J. R.(Editors) Aspergillus: 50 years on. Progress in industrial microbiologyvol 29. Elsevier Amsterdam 1994. pp. 641-666). Gene expression infilamentous fungi has been reviewed in Punt et al. (2002) TrendsBiotechnol 2002 May; 20(5):200-6, Archer & Peberdy Crit. Rev Biotechnol(1997) 17 (4):273-306.

Transformed Yeast

In another embodiment, the transgenic organism can be a yeast.

A review of the principles of heterologous gene expression in yeast areprovided in, for example, Methods Mol Biol (1995), 49:341-54, and CurrOpin Biotechnol (1997) October; 8 (5):554-60

In this regard, yeast—such as the species Saccharomyces cereviseae orPichia pastoris (see FEMS Microbiol Rev (2000 24 (1):45-66), may be usedas a vehicle for heterologous gene expression.

A review of the principles of heterologous gene expression inSaccharomyces cerevisiae and secretion of gene products is given by EHinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression ofheterologous genes”, Yeasts, Vol 5, Anthony H Rose and J StuartHarrison, eds, 2nd edition, Academic Press Ltd.).

For the transformation of yeast, several transformation protocols havebeen developed. For example, a transgenic Saccharomyces according to thepresent invention can be prepared by following the teachings of Hinnenet al., (1978, Proceedings of the National Academy of Sciences of theUSA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, Het al (1983, J Bacteriology 153, 163-168).

The transformed yeast cells may be selected using various selectivemarkers—such as auxotrophic markers dominant antibiotic resistancemarkers.

Transformed Plants/Plant Cells

A host organism suitable for the present invention may be a plant. Areview of the general techniques may be found in articles by Potrykus(Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou(Agro-Food-Industry Hi-Tech March/April 1994 17-27).

Culturing and Production

Host cells transformed with the nucleotide sequence of the presentinvention may be cultured under conditions conducive to the productionof the encoded enzyme and which facilitate recovery of the enzyme fromthe cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in questions and obtaining expressionof the enzyme.

The protein produced by a recombinant cell may be displayed on thesurface of the cell.

The enzyme may be secreted from the host cells and may conveniently berecovered from the culture medium using well-known procedures.

Secretion

Often, it is desirable for the enzyme to be secreted from the expressionhost into the culture medium from where the enzyme may be more easilyrecovered. According to the present invention, the secretion leadersequence may be selected on the basis of the desired expression host.Hybrid signal sequences may also be used with the context of the presentinvention.

Typical examples of heterologous secretion leader sequences are thoseoriginating from the fungal amyloglucosidase (AG) gene (glaA—both 18 and24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeastse.g. Saccharomyces, Kluyveromyces and Hansenula) or the α-amylase gene(Bacillus).

By way of example, the secretion of heterologous proteins in E. coli isreviewed in Methods Enzymol (1990) 182:132-43.

Detection

A variety of protocols for detecting and measuring the expression of theamino acid sequence are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic and amino acidassays.

A number of companies such as Pharmacia Biotech (Piscataway, N.J.),Promega (Madison, Wis.), and US Biochemical Corp (Cleveland, Ohio)supply commercial kits and protocols for these procedures.

Suitable reporter molecules or labels include those radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. No. 3,817,837;U.S. Pat. No. 3,850,752; U.S. Pat. No. 3,939,350; U.S. Pat. No.3,996,345; U.S. Pat. No. 4,277,437; U.S. Pat. No. 4,275,149 and U.S.Pat. No. 4,366,241.

Also, recombinant immunoglobulins may be produced as shown in U.S. Pat.No. 4,816,567.

Fusion Proteins

The amino acid sequence for use according to the present invention maybe produced as a fusion protein, for example to aid in extraction andpurification. Examples of fusion protein partners includeglutathione-S-transferase (GST), 6×His (SEQ ID NO: 33), GAL4 (DNAbinding and/or transcriptional activation domains) and(β-galactosidase). It may also be convenient to include a proteolyticcleavage site between the fusion protein partner and the proteinsequence of interest to allow removal of fusion protein sequences.

Preferably, the fusion protein will not hinder the activity of theprotein sequence.

Gene fusion expression systems in E. coli have been reviewed in CurrOpin Biotechnol (1995) 6 (5):501-6.

In another embodiment of the invention, the amino acid sequence may beligated to a heterologous sequence to encode a fusion protein. Forexample, for screening of peptide libraries for agents capable ofaffecting the substance activity, it may be useful to encode a chimericsubstance expressing a heterologous epitope that is recognised by acommercially available antibody.

Large Scale Application

In one preferred embodiment of the present invention, the amino acidsequence is used for large scale applications.

Preferably the amino acid sequence is produced in a quantity of from 1 gper litre to about 2 g per litre of the total cell culture volume aftercultivation of the host organism.

Preferably the amino acid sequence is produced in a quantity of from 100mg per litre to about 900 mg per litre of the total cell culture volumeafter cultivation of the host organism.

Preferably the amino acid sequence is produced in a quantity of from 250mg per litre to about 500 mg per litre of the total cell culture volumeafter cultivation of the host organism.

Food

The composition of the present invention may be used as—or in thepreparation of—a food. Here, the term “food” is used in a broadsense—and covers food for humans as well as food for animals (i.e. afeed). In a preferred aspect, the food is for human consumption.

The food may be in the form of a solution or as a solid—depending on theuse and/or the mode of application and/or the mode of administration.

Food Ingredient

The composition of the present invention may be used as a foodingredient.

As used herein the term “food ingredient” includes a formulation, whichis or can be added to functional foods or foodstuffs and includesformulations which can be used at low levels in a wide variety ofproducts that require, for example, acidifying or emulsifying.

The food ingredient may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

Food Products

The composition of the present invention can be used in the preparationof food products such as one or more of: confectionery products, dairyproducts, meat products, poultry products, fish products and bakeryproducts.

The present invention also provides a method of preparing a food or afood ingredient, the method comprising admixing a lipolytic enzymeaccording to the present invention with another food ingredient.

Further preferable aspects are presented in the accompanying claims andin the following Figures and examples.

FIG. 1 shows PCR fragment SEQ ID No. 1, which is a partial non-enzymeencoding polynucleotide; this sequence is a ribosomal 16S RNA genewidely used for taxonomic comparisons;

FIG. 2 shows PCR fragment SEQ ID No. 2, which is a partial non-enzymeencoding polynucleotide; this sequence is a ribosomal 16S RNA genewidely used for taxonomic comparisons;

FIG. 3 shows a polynucleotide encoding a lipolytic enzyme according tothe present invention (SEQ ID No. 3);

FIG. 4 shows an amino acid sequence of a lipolytic enzyme according tothe present invention (SEQ ID No. 4);

FIG. 5 shows the structure of the lipolytic enzyme expression vectorspGTK(L131) and pET11(131-51);

FIG. 6 shows a graph of the effect of a lipolytic enzyme fromStreptomyces sp. L130 on digalactosyldiglyceride in dough;

FIG. 7 shows in graphical form the effect of a lipolytic enzyme fromStreptomyces sp. L131 on digalactosyldiglyceride in dough;

FIG. 8 shows in graphical form the effect of a lipolytic enzyme fromStreptomyces on triglyceride in dough;

FIG. 9 shows the pH profile of the lipolytic enzyme obtained fromStreptomyces sp. L131 on galactolipid substrate;

FIG. 10 shows a TLC plate of lipids extracted from dough treated with alipolytic enzyme from Streptomyces expressed in E. coli labelled #236;Lane 1=control; Lane 2=#236, 0.225PLU-7/g flour; Lane 3=#236, 0.45PLU-7/g flour; Lane 4=#236, 0.675 PLU-7/g flour; Lane 5=DGDG referencematerial.

FIG. 11 shows the construction of expression vector pRX487 frompUC18(L131R) and pIJ48;

FIG. 12 shows in graphical form the effect of temperature on stabilityand activity of a lipolytic enzyme from Streptomyces sp L131;

FIG. 13 shows in graphical form the substrate specificities ofgalactolipases from Streptomyces sp L131, Streptomyces avermitillis,Corynebacterium efficiens and Thermobifida fusca;

FIG. 14 shows the structure of an expression vector pCB5(TF) forexpression of Thermobifida fusca lipase in C. glutamicum;

FIG. 15 shows a sequence alignment of L131 (SEQ ID NO: 4) and homologuesS. avermitilis (SEQ ID NO: 14) and T. fusca (Residues 177-548 of SEQ IDNO: 5). Figure also discloses a consensus sequence disclosed as SEQ IDNO: 30);

FIG. 16 shows a HPTLC plate of reaction products from enzyme treatmentof crude soya oil samples. Lane 1=control, Lane 2=99% crude oil and 1%K371 10% in water, Lane 3=98% crude oil and 2% K371 10% in water, Lane4=97% crude oil and 3% K371 10% in water, Lane 5=99.7% crude oil and0.3% Lecitase Ultra™ #3108 1% in water, Lane 6=99% crude oil, 0.3%Lecitase Ultra™ #3108 1% in water and 0.7% water. As referencephoshpatidylcholine (PC) is analysed; and

FIG. 17 shows a HPTLC plate of reaction products from enzyme treatmentof crude soya oil samples. Lane 1=control, Lane 2=99% crude oil and 1%K371 10% in water, Lane 3=98% crude oil and 2% K371 10% in water, Lane4=97% crude oil and 3% K371 10% in water, Lane 5=99.7% crude oil and0.3% Lecitase Ultra™ #3108 1% in water, Lane 6=99% crude oil, 0.3%Lecitase Ultra™ #3108 1% in water and 0.7% water, together withreference lanes of cholesterol ester, monoglyceride, diglyceride,triglyceride and plant sterol.

Example 1 Identification of a Galactolipase Producing Bacterial Strain

Two microbial strains with a similar phenotype coded L130 and L131 wereisolated from soil collected in Southern Finland. The 16s RNA genes ofthese two strains were 10 amplified by standard PCR usingoligonucleotide primers 536f (CAGCMGCCGCGGTAATWC) (SEQ ID NO: 18) and1392r-primer (ACGGGCGGTGTGTRC) (SEQ ID NO: 19). The resulting PCRfragments were partially sequenced. SEQ ID Nos. 1 and 2 are non-enzymeencoding polynucleotides. These sequences are ribosomal 16s RNA geneswidely used for taxonomic comparisons. SEQ ID No. 1 and SEQ ID No. 2were found 15 to have a high similarity. The sequences were thencompared to the 16s RNA gene sequences in GenBank. For both isolates thehighest homology (97%) was observed with the sequence of a 16s RNA genefrom Streptomyces thermosacchari Thus, the strains were namedStreptomyces sp. L130 and Streptomyces sp. L131.

Example 2 Preparation of Lipolytic Enzyme (Galactolipase) Samples fromStrains Streptomyces sp. L130 and L131

0.5 l of LB medium was inoculated with Streptomyces L130 and cultivatedon a rotary shaker at 200 rpm and 30° C. for 2 days. This culture wasused as inoculum for a 10 l fermentor containing the same medium. Thecultivation was continued for 3 days at 30° C., 600 rpm stirring rateand 0.5 v/v aeration. The fermentation broth was cleared bycentrifugation (15 min at 5000 rpm) and Triton X-100 was added to finalconcentration of 0.1%. The solution was concentrated using Vivaflow 200ultrafiltration cell (Vivascience AG, Hannover, Germany) to 300 ml. Theconcentrate was dialysed against 10 l of 20 mM Tris HCl buffer, pH 7containing 2 mM CaCl₂ and 2 mM MgCl₂ followed by dialysis against 0.5 1ml of 85% glycerol. The resulting preparations contained 90 U ofgalactolipase activity assay as defined above (GLU-7). The strainStreptomyces L131 was cultivated under the same conditions and itsculture broth was concentrated by the same procedure. The resultinggalactolipase preparation contained 70 U of activity.

Example 3 Baking Experiments

The galactolipases from bacterial isolates L130 and L131 indicated ahigh activity on polar lipid substrates, galactolipids (DGDG) andphospholipids, (galactolipase and phospholipase activity), equivalent tothat of a Fusarium oxysporum lipase (Lipopan F™ Novozymes A/S Denmark):however the galactolipase from bacterial isolates L130 and L131 (i.e.the lipolytic enzyme according to the present invention) were found tohave no significant activity of triglycerides. This contrasts sharplywith the activity Fusarium oxysporum lipase—LipopanF™.

The lipolytic enzymes from bacterial isolates L130 and L131 wereprepared as described in Example 2 and were analysed forcharacterisation of their activity on glycolipids, phospholipids andtriglycerides, both in standard assay conditions and within a dough.

Small scale baking experiments and a model dough system. Both enzymesare very active on galactolipids in flour.

Materials and Methods

Three samples of each enzyme were prepared as in Example 3. Each samplewas labelled as shown in table 1:

TABLE 1 ID Organism Label GLU-7 PLU-7 180 Streptomyces Lipolytic enzyme0.95 1.31 spp L 130 A 0.58 PLU/mL 181 Streptomyces Lipolytic enzyme 0.911.31 spp L 130 B 0.44 PLU/mL. 182 Streptomyces Lipolytic enzyme 1.211.53 spp L 130 C 1.8 PLU/mL. 183 Streptomyces Lipolytic enzyme 0.63 1.29spp L 131 A 0.54 PLU/mL. 184 Streptomyces Lipolytic enzyme 0.84 1.16 sppL 131 B 0.64 PLU/mL. 185 Streptomyces Lipolytic enzyme 1.35 1.17 spp L131 C 0.85 PLU/mL.

The phospholipase and galactolipase activity of the enzymes wereassessed using the phospholipase activity assay (PLU-7) and thegalactolipase activity assay (GLU-7) mentioned herein above.

Dough Slurry Experiment

0.8 gram Wheat flour was scaled in a 12 ml centrifuge tube with lid. 1.5ml water containing the enzyme was added. The sample was mixed on aWhirley and placed in a heating cabinet at 30° C. for 60 minutes. 6 mln-Butanol:Ethanol 9:1 was added, and the sample was mixed again untilthe flour was finely distributed in the solvent. The tubes were theplaced in a water bath at 95° C. for 10 minutes. Then mixed again andplaced on a rotation device 45 rpm, for 45 minutes.

The sample was then centrifuged at 2000 g for 10 minutes. And 2 mlsupernatant was transferred to a 10 ml dram glass. The solvent wasevaporated at 70° C. under a steam of nitrogen.

The isolated lipids are analysed by GLC.

Gas Chromatography

Perkin Elmer 8420 Capillary Gas Chromatography equipped with WCOT fusedsilica column 12.5 m×0.25 mm ID×0.1 μm 5% phenyl-methyl-silicone (CP Sil8 CB from Crompack).

Carrier: Helium.

Injection: 1.5 μL with split.

Detector: FID. 385° C.

Oven program: 1 2 3 4 Oven temperature [° C.] 80 200 240 360 Isothermal,time [min] 2 0 0 10 Temperature rate [° C./min] 20 10 12Sample preparation: Lipid extracted from 0.2 gram flour was dissolved in2 mL heptane:pyridine 2:1 containing an internal standard heptadecane, 2mg/mL. 500 μL of the sample was transferred to a crimp vial. 100 μLMSTFA (N-Methyl-N-trimethylsilyl-trifluoracetamid) was added and thereaction incubated for 15 minutes at 90° C.Calculation: Response factors for monoglycerides, diglycerides,triglycerides, free fatty acid and galactolipids were determined fromreference mixtures of these components. Based on these response factorsthe lipids in the dough were calculated.

Results.

The samples of enzyme from Streptomyces were analyzed for phospholipaseand galactolipase activity with results shown in table 2. The activityratio PLU-7/GLU-7 was also calculated. The mean ratio for the sampleswas 1.4, but with some deviation in some of the samples, which might beexplained by analytical deviations.

TABLE 2 Sample ID Organism GLU-7 PLU-7 Ratio PLU-7/GLU-7 180 L 130 A0.95 1.31 1.4 181 L 130 B 0.91 1.31 1.4 182 L 130 C 1.21 1.53 1.3 183 L131 A 0.63 1.29 2.0 184 L 131 B 0.84 1.16 1.4 185 L 131 C 1.35 1.17 0.9

Dough Experiment.

The activity of the enzyme on wheat lipids was tested in the doughslurry experiment as mentioned under materials and Methods. The isolatedlipids from the dough were analysed by GLC as shown in table 3

TABLE 3 GLC analysis of dough lipids (% based on flour weight). Enzymedosage Sample PLU/g ID flour FFA MGMG DGMG MGDG DGDG TRI 185 0.1050.1642 0.0042 0.0380 0.0345 0.1520 0.5515 185 0.263 0.1687 0.0130 0.06700.0239 0.0941 0.5470 185 0.526 0.2096 0.0121 0.0664 0.0158 0.0617 0.5460185 1.05 0.2597 0.0036 0.0546 0.0068 0.0303 0.5301 182 0.097 0.15420.0051 0.0563 0.0313 0.1148 0.5475 182 0.244 0.1687 0.0159 0.0785 0.02000.0566 0.5280 182 0.488 0.2095 0.0055 0.0646 0.0098 0.0219 0.5418 1820.976 0.2581 0.0092 0.0439 0.0043 0.0045 0.5579 Control 0 0.1529 0.00060.0188 0.0440 0.1443 0.5054 Lipopan 1.47 0.23 0.03 0.10 0.01 0.07 0.44F ™ FFA = free fatty acids. MGMG = monogalactosylmonoglyceride. DGMG =digalactosyldiglyceride. MGDG = monogalactosyldiglyceride. DGDG =digalactosyldiglyceride. TRI = triglyceride.

The results from table 3 and table 4 confirm that the enzymes isolatedin the supernatant from fermentation of Streptomyces sp L130 and L131are very active on galactolipids in a dough. The diesters DGDG and MGDGare hydrolyzed to the corresponding monoesters DGMG and MGMG. Theresults are also illustrated graphically in FIGS. 6 and 7. These resultsconfirm that both enzymes are very active at low dosage 0-0.2 Units/gflour and corresponding amount of monoester is produced. At higherdosage 0.4-1 Units/gram flour DGDG is further degraded but also somehydrolysis of the monoesters are observed. This may indicate the enzymesare not specific to the position of the fatty acid in the galactolipidmolecule.

The activity of the enzymes on triglyceride, as illustrated in FIG. 8,is almost not existent. It is therefore concluded that the enzymestested have no significant effect on triglyceride. This is also inagreement with some experiments conducted on tributyrin as substrate,where no activity was observed.

SUMMARY

A lipolytic enzyme was isolated in the supernatant from fermentation ofStreptomyces sp.

The lipolytic enzyme was found to have both phospholipase andgalactolipase activity, but no significant activity on triglycerides.The ratio of phospholipase:galactolipase activity was approx. 1.4 forthe samples tested.

Dough slurry experiments confirms that the enzymes were active ongalactolipids in the flour. The enzymes were active in dough at a verylow dosage 0-0.2 Units/g flour. Commercial phospholipases like LipopanF™ (Novozymes A/S, Denmark) need to be dosed in 3-4 times higher dosagein order to obtain the same effect on galactolipids. The dough slurryexperiments also confirmed that the enzymes from Streptomyces sp. had nomeasurable activity on triglycerides.

Example 4 Cloning of the Lipolytic Enzyme Gene from Streptomyces sp.L131

The chromosomal DNA was isolated from Streptomyces sp. L131 using amodification of a standard method. Bacteria were grown on a rotaryshaker in LB medium at 30° C. and high aeration (100 ml of medium per0.51 baffled flask, 200 rpm) to early stationary phase. From 500 mlbacterial culture cells were collected with centrifugation and washedonce with lysis buffer (550 mM glucose, 100 mM Tris, 2 mM EDTA, pH 8.0).

Cell pellet was re-suspended in 10 ml of lysis buffer and lysozyme wasadded to 1 mg/ml. Cells were incubated at 37° C. for at least 15 min.The progress of lysozyme digestion was followed by transferring aliquotsof bacterial suspension into 1% SDS solution and measuring theabsorption of the resulting mixture at 600 nm. The amount of lysozymeand incubation time were adjusted so that at least 70-90% of all cellswere lysed as evidenced by the decrease in A₆₀₀. At this point of time,SDS was added to the bacterial suspension to 1% and proteinase K to 0.1mg/ml. The suspension was incubated at 56° C. for 30 min followed byextractions with phenol and chloroform. After chloroform extraction, DNAwas precipitated with sodium acetate (0.5M final concentration) andisopropanol (0.6 vol/vol) and the DNA pellet was washed with 70%ethanol, dried in vacuum and dissolved in TE buffer (10 mM Tris, 1 mMEDTA) containing RNAse A (0.01 mg/ml).

The DNA was partially digested with restriction endonuclease Sau3A andthe hydrolysates fractionated on a 0.8% agarose gel. The 3-10 kbfraction of the Sau3A was isolated from agarose gels by electroelution.This DNA preparation was used to construct a gene library usingStratagene's (LaJolla, USA) ZAP Express/Predigested Vector/GigapackCloning Kit (product #239615). Ligation, packaging, amplification oflibrary and its conversion to the phagemid form were carried outaccording to the protocols provided by Stratagene. Plasmid form of theresulting gene library was screened on indicator plates prepared asfollows. 80 ml of sterile LB agar containing 25 mg/l of kanamycin wasplaced into each 15 cm Petri dish and allowed to solidify. Subsequently,10 ml top agar layer was added containing 0.5% DGDG and 0.0005%Safranine O. The gene library was plated at a density of approximately5000 colonies per 15 cm plate. The plates were incubated at 37° C. for24 h followed by a four-day incubation at room temperature. A cloneforming red halo on indicator plate was selected from the library andpurified by cloning on a new indicator plate.

The plasmid isolated from this clone (named pBK(L131)) was used tore-transform E. coli strain XL1-Blue MRF′ to kanamycin resistance. Allsuch transformants displayed galactolipase-positive phenotype. pBK(L131)contained an approximately 7.5 kb insert. This insert was sequenced. Onesequenced region (SEQ ID No. 3) was found to contain an open readingframe encoding a protein (SEQ ID No. 4) showing homology to a knownlipase from Streptomyces rimosus. This lipase, a member of so-calledGDSX (SEQ ID NO: 31) family of lipases/esterases/acyl transferases isonly known to be able to hydrolyse neutral lipids and artificial lipasesubstrates.

A series of deletions and sub-clones of the original insert wereconstructed and tested for galactolipase activity. It was found that adeletion derivative carrying 3 kb EcoRI—SacI fragment of the originalinsert still retains full DGDGse activity. This data correlated wellwith the results of partial DNA. One area demonstrated homology to knownlipases. This area was subsequently sequenced completely. Comparison ofthis sequence with the GenBank revealed that the closest homologue(58.5%) of the L131 galactolipase that has been biochemicallycharacterised is a lipase from S. rimosus, and identified as alipid:acyl transferase in WO04/064987 and WO04/064537.

Expression of L131 Galactolipase in E. coli.

The standard pET-system, in which the gene is under control of the T7phage promoter, was used in to express the L131 galactolipase in E.coli.

Expression of L131 Galactolipase in Streptomyces lividans.

The shuttle vector pRX487-5 (FIG. 11) (derived from pIJ4987: Kieser T.et al Practical Streptomyces genetics. The John Innes Foundation,Crowes, Norwich, England (2000)) used for expression of L131galactolipase in S. lividans combines E. coli plasmid pUC18 and the S.lividans plasmid IJ487. In pRX487-5, the lac promoter of pUC18 is placedupstream of promoter-less kanamycin phosphotransferase gene of pIJ487.Indeed, the plasmid transformed E. coli not only to ampicillin but alsoto at least a low level (5 mg/l) of kanamycin resistance. The vectorcontains unmodified EcoRI-XbaI fragment of the chromosomal DNA of S.thermosacchari comprising the complete coding sequence of thegalactolipase gene, about 160 bp of upstream non-coding sequence and 420bp of downstream non-coding sequence. All transformants displayedsimilar levels of galactolipase activity as judged by the halo formationon indicator plates. Similarly, when S. lividans carrying pRX487-5 wascultivated at 10 l level and the resulting culture was cloned onindicator plates, all clones appeared to produce equal amounts ofgalactolipase activity. In small shake-flask cultures inoculated byvegetative cells directly from plates, the transformants typicallyproduced about 10-20 mU/ml of galactolipase activity after 3 days ofcultivation. When shake-flask cultures were inoculated with spores ofthe recombinant S. lvividans, higher galactolipase activities weremeasured (about 30 mU/ml), correlating with higher biomass accumulation.In an experiment where S. lividans carrying pRX487-5 was grown infermentor under high aeration conditions and fed-batch mode (seematerial and methods for details) biomass accumulation reached 170 g/l(wet weight). Accordingly, much higher galactolipase activity—about 1U/ml was detected.

Biochemical Properties of L131.

Some biochemical properties of L131 were tested. The pH optimum of theenzyme was found to be around 6.5-7.5 (FIG. 9). The enzyme exhibitedmaximum activity towards DGDG at a temperature ˜50° C. Above thistemperature inactivation occurred, but not sharply, and after 20 minincubation in 90° C., ˜10% of residual activity was detected (FIG. 12).

Example 5 Expression of Streptomyces L131 Lipolytic Enzyme According tothe Present Invention Gene in E. coli

The open reading frame of pBK(L131) encoding presumptive lipolyticenzyme according to the present invention was amplified by PCR usingprimers oL131-5 (GGTGAATT′CATGAGATTGACCCGATCCCTGTCGG, sense primer) (SEQID NO: 20) and oL131-3 (ACTTCTAGAGCGGCGCCACCGTGACGTACA, anti-senseprimer) (SEQ ID NO: 21). The amplified DNA fragment was digested withEcoRI and XbaI and cloned into a B. subtilis-E. coli shuttle vectorpGTK44. This vector has been constructed by substituting the SalI-EcoRIfragment of plasmid pGTK44 (Povelainen et al., Biochem J. 371, 191-197(2003)) containing degQ36 promoter with EcoRI-SalI fragment of pGT44(Kerovuo J. et al. Biotechnology Letters 22, 1311-1317 (2000)).

Galactolipase activity was detected in E. coli transformed with theresulting plasmid pGTK44(L131) (FIG. 5) using indicator plates. Controltransformants (containing parent plasmid pGTK44) weregalactolipase-negative. Thus, protein sequence represented by SEQ ID No4 indeed possesses galactolipase activity. The same pair of primersamplified a fragment of the same size (by agarose gel electrophoresis)with chromosomal DNA of Streptomyces sp. L130 further confirming earlierobservations about close similarity of the two isolated strains andtheir galactolipase genes.

For expression in E. coli under control of the T7 phage promoter, thededuced galactolipase coding region was amplified by PCR usingchromosomal DNA of the Streptomyces sp. L131 as template and the twooligonucleotide primers (oL131-51 GGTCATGCTAGCATGAGATTGACCCGATCCCTGTCGG(SEQ ID NO: 22) and oL131-31 GCATGGATCCGCGGCGCCACCGTGACGTACA) (SEQ IDNO: 23). The PCR product was digested with NheI and BamHI and ligatedwith pET11a (Novagen, USA) vector digested with the same restrictionendonucleases. The ligation mixture was used to transform the E. colistrain XL-Blue1 MRF′ and 12 different plasmid clones with restrictionpatterns corresponding to the structure of pET11(131-51) (FIG. 4) wereisolated. Each plasmid clone was used to separately transform the E.coli strain BL21(DE3) and the resulting transformants were grown onLB-ampicilin containing galactolipase activity indicator layer (Example4). Most clones did express active galactolipase. One clone(pET11(131-51)-12) was selected as a source of recombinant galactolipasefor subsequent characterisation.

The enzyme expressed in E. coli (labelled #236) was analysed and foundto have: 0.33 GLU/ml and 0.36 PLU/ml, when analysed using the GLU-7assay and PLU-7 assay taught herein.

In liquid culture E. coli BL21(DE3) expressed about 2 mU/ml ofgalactolipase activity after 40 h cultivation in LB-ampicillin broth(37° C., 200 rpm shaking). Essentially all of the activity was found inthe culture broth. No galactolipase activity was detected in E. coliBL21(DE3) transformed with pET11a (Novagen, USA) and cultivated underthe same conditions.

About four litres of galactolipase-containing culture broth culture wasconcentrated on a rotary evaporator to about 300 ml and dialysed against15 l of 20 mM Tris HCl buffer, pH 7 containing 2 mM CaCl₂ and 2 mMMgCl₂. The dialysed material was again concentrated on a rotaryevaporator to about 30 ml and dialysed against 2 l of 50% glycerol. Theresulting preparation (18 ml) contained about 100 mU/ml of galactolipaseactivity.

The enzyme expressed in E. coli (labelled #236) was also tested indough. High activity on galactolipids was observed in dough as can beseen from FIG. 10, which shows a TLC plate.

Example 6 Expression of the Lipolytic Enzyme According to the PresentInvention Gene from Streptomyces sp. L131 in Different Hosts

Construction of the vector pGTK44(L131) has been outlined in the Example5. Besides E. coli, this vector can be used to produce Streptomyces L131lipolytic enzyme according to the present invention in Bacillus. Usingthis vector is only one of many possible ways to express the L131lipolytic enzyme according to the present invention in Bacillus. Forexample, the pst promoter employed in pGTK44(L131) may be replaced byany other strong constitutive or regulated promoter active in Bacillus.Many such promoters are known in the art. For example, degQ36 promoter(Yang M et al. J. Bacteriol. 166, 113-119 (1986)), cdd promoter, alsoknown as p43 (Wang P Z, Doi R H. J. Biol. Chem. 259, 8619-8625 (1984),amylase or neutral protease promoters etc. In addition to pGTK44(L131)and other Bacillus vectors based on pTZ12 replicon (Aoki T. et al., Mol.Gen. Genet. 208, 348-352 (1987)) any other plasmid vector (e.g pUB110,Gryczan T J et al. J. Bacteriol. 134, 318-29 (1978) and its derivatives)can be used.

Other preferred hosts for expression of the Streptomyces L131 lipolyticenzyme according to the present invention gene are high-GC Gram positivebacteria, in particular, Streptomyces, (for example, S. lividans, S.coelicolor, S. griseus, S. natalensis, S. rubiginosus, S. olivaceus, S.olivochromogenes, S. violaceoruber), In such hosts, the lipolytic enzymeaccording to the present invention gene can be introduced under its ownpromoter on a multi-copy vector (e.g. using pIJ110 derivatives such aspIJ486, Ward et al. Mol. Gen. Genet. 203, 468-478 (1986)) or placedunder control of a strong Streptomyces promoter, for example ermE*(Schmitt-John T, Engels J W. Appl. Microbiol. Biotechnol. 36, 493-498(1992)) or thiostreptone-inducinbe tipA promoter (Kieser T et al. inPractical Streptomyces Genetics, p. 386, The John Innes Foundation,Norwich UK (2000)).

In addition to prokaryotic hosts, L131 lipolytic enzyme gene may beexpressed in one of the many suitable fungal species. In particular,yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, Hansenula polymorpha are suitable. In yeast, thelipolytic enzyme gene may be placed under control of any of the knownstrong yeast promoters, such glycolytic promoters (PGK, GDH, ENO etc)phosphate starvation induced promoters such as PHO5, the promoters ofethanol/methanol metabolism such as ADH1 promoter in S. cerevisiae ormethanol-inducible promoters in H. polymorpha or P. pastoris.

When expressing the lipolytic enzyme gene in any host, construction of asynthetic or semi-synthetic gene encoding the sequence of SEQ ID 4 wouldbe advantageous. Likewise, partly or completely synthetic genes may bedesigned based on sequences available through homology searches insilico as explained in Example 4. Such sequences, may incorporate anumber of useful features that are absent in wild-type lipolytic enzymegenes. For, example, the codon bias can be corrected to bettercorrespond codon preferences of the expression hosts. One special caseof codon bias correction useful for all hosts is to convert the GTGinitiation codon of SEQ ID No 3 into ATG. Another typical modificationobvious for a man skilled in the art is to exchange the nativeStreptomyces signal sequence of the L131 lipolytic enzyme with a signalsequence native to or known to be functional in the chosen expressionhost.

Previous examples of useful expression systems for L131 lipolytic enzymefocused on using plasmid vectors for the introduction of the lipolyticenzyme gene into the expression host. This is indeed the preferred modeto implement current invention. However, an alternative approach ofintegrating the expression cassette (including promoter, lipolyticenzyme gene coding region and an optional transcription terminator) intoa chromosome is also feasible. In particular, multi-copy integration ofthe expression cassette into the host chromosome would be efficient.

The recombinant hosts expressing the lipolytic enzyme gene can be,advantageously, mutated to reduce the level of protease activity in theculture broth. The cultivation of any of such recombinant hosts can becarried out in the presence of compounds stabilising the enzyme. Suchcompounds may be various proteins (e.g. casein, peptone of serumalbumin) or different lipids, lysolipids or detergents (e.g.galactolipids, mono- and diacylglycerols or Triton X-100).

Example 7 Acyl-Transferase Activity of Streptomyces L131 LipolyticEnzyme and its Derivatives

Some lipases may also possess acyl-transferase activity. In particular,some members of the GDSX (SEQ ID NO: 31) family, for example, Aeromonashydrophila acyltransferase (P10480) (taught in copending InternationalApplication No. PCT/IB2004/000655) have high acyl-transferase activity.Thus, Streptomyces L131 lipolytic enzyme may be predicted to have alsothe acyl-transferase activity as well. This activity can be furtherenhanced through random mutagenesis/directed evolution. Moreover, sinceA. hydrophila acyl-transferase and Streptomyces L131 lipolytic enzymeshare the same overall protein fold, combining the substrate specificityof Streptomyces L131 lipolytic enzyme with high transferase efficiencyof the Aeromonas enzyme is possible. This combination may be achievedthrough the known techniques of targeted mutagenesis/protein design orby gene shuffling.

Example 8 Identification of Alternative Lipolytic Enzymes from OtherStreptomyces Species

The GDSX (SEQ ID NO: 31) family of esterase's (Upton C, Buckley J T.Trends Biochem. Sic. 20, 178-179 (1995), pfam00657.11) is a group ofesterases/lipases/acyl transferases sharing a specific sequence motifaround the active site serine (GDSX (SEQ ID NO: 31) where X is ahydrophobic amino acid residue). This group of enzymes is also known aslipase family 11 (Arpigny J L, Jaeger K-E. Biochem. J. 343, 177-183(1999)). Although this family includes many different types ofesterases, lipases and acyl-transferases, the lipolytic enzyme accordingto the present invention is a GDSX (SEQ ID NO: 31) enzyme.

Thus, the sequences taught in the present invention of the Streptomycessp. L131 lipolytic enzyme (galactolipase) can be used in silico toidentify other galactolipases from other species of Streptomyces.

To determine if a protein has the GDSX (SEQ ID NO: 31) motif accordingto the present invention, the sequence is preferably compared with thehidden markov model profiles (HMM profiles) of the pfam database.

Pfam is a database of protein domain families. Pfam contains curatedmultiple sequence alignments for each family as well as profile hiddenMarkov models (profile HMMs) for identifying these domains in newsequences. An introduction to Pfam can be found in Bateman A et al.(2002) Nucleic Acids Res. 30; 276-280. Hidden Markov models are used ina number of databases that aim at classifying proteins, for review seeBateman A and Haft D H (2002) Brief Bioinform 3; 236-245.

For a detailed explanation of hidden Markov models and how they areapplied in the Pfam database see Durbin R, Eddy S, and Krogh A (1998)Biological sequence analysis; probabilistic models of proteins andnucleic acids. Cambridge University Press, ISBN 0-521-62041-4. TheHammer software package can be obtained from Washington University, StLouis, USA.

Alternatively, the GDSX (SEQ ID NO: 31) motif can be identified usingthe Hammer software package, the instructions are provided in Durbin R,Eddy S, and Krogh A (1998) Biological sequence analysis; probabilisticmodels of proteins and nucleic acids. Cambridge University Press, ISBN0-521-62041-4 and the references therein, and the HMMER2 profileprovided within this specification.

The PFAM database can be accessed, for example, through several serverswhich are currently located at websites maintained by the SangerInstitute (UK) in conjunction with Wellcome Trust Institute, theInstitut National de la Recherche Agronomique, and the Center forGenomics and Bioinformatics of the Karolinska Institutet, among others.

The database offers a search facility where one can enter a proteinsequence. Using the default parameters of the database the proteinsequence will then be analysed for the presence of Pfam domains. TheGDSX (SEQ ID NO: 31) domain is an established domain in the database andas such its presence in any query sequence will be recognised. Thedatabase will return the alignment of the Pfam00657 consensus sequenceto the query sequence.

Preferably when aligned with the Pfam00657 consensus sequence thelipolytic enzyme for use in the compositions/methods of the inventionhave at least one, preferably more than one, preferably more than two,of the following, a GDSx (SEQ ID NO: 31) block, a GANDY (SEQ ID NO: 35)block, a HPT block. Suitably, the lipolytic enzyme may have a GDSx (SEQID NO: 31) block and a GANDY (SEQ ID NO: 35) block. Alternatively, theenzyme may have a GDSx (SEQ ID NO: 31) block and a HPT block. Preferablythe enzyme comprises at least a GDSx (SEQ ID NO: 31) block.

The pfam00657 GDSX (SEQ ID NO: 31) domain is a unique identifier whichdistinguishes proteins possessing this domain from other enzymes.

In addition or as an alternative thereto, alternative lipolytic enzymesfrom other Streptomyces species can be identified by conducting asequence identity comparison and/or hybridisation with one or more ofthe PCR sequence fragments shown as SEQ ID No. 1 or SEQ ID No. 2.Suitably, the comparisons may be carried out with fragments comprisingover 15 nucleotides of SEQ ID No. 1 or SEQ ID No. 2, preferably withfragments comprising over 20 nucleotides of SEQ ID No. 1 or SEQ ID No.2. Suitably, the complete sequences shown as SEQ ID No. 1 or SEQ ID No.2 could be used. Preferably, the hybridisation is carried out at high orvery high stringency conditions. Nucleotide sequences having at least80%, preferably at least 85%, at least 90%, at least 95%, at least 97%,at least 98% or at least 99% identity to SEQ ID No. 1 or SEQ ID No. 2indicate strains of Streptomyces which may be sources of the lipolyticenzyme, i.e. the galactolipase, according to the present invention.

Example 9 Identification of Galactolipases for Use in the Methods andUses of the Present Application

As mentioned above, the sequence of the novel Streptomycesthermosacchari L131 offers for the possibility for in silicoidentification of new family II galactolipases. In this regard, oneparticular region which may be of particular interest is the GDSX (SEQID NO: 31) motif.

The GDSX (SEQ ID NO: 31) motif is comprised of four conserved aminoacids. Preferably, the serine within the motif is a catalytic serine ofthe lipid acyltransferase enzyme. Suitably, the serine of the GDSX (SEQID NO: 31) motif may be in a position corresponding to Ser-16 inAeromonas hydrophila lipolytic enzyme taught in Brumlik & Buckley(Journal of Bacteriology April 1996, Vol. 178, No. 7, p 2060-2064).

To determine if a protein has the GDSX (SEQ ID NO: 31) motif, thesequence is preferably compared with the hidden markov model profiles(HMM profiles) of the pfam database. As mentioned in Example 8, pfam isa database of protein domain families. Thus, the pfam database may alsobe used to identify suitable enzymes from genera other thanStreptomyces.

Alternatively, the GDSX (SEQ ID NO: 31) motif can be identified usingthe Hammer software package, the instructions are provided in Durbin R,Eddy S, and Krogh A (1998) Biological sequence analysis; probabilisticmodels of proteins and nucleic acids. Cambridge University Press, ISBN0-521-62041-4 and the references therein, and the HMMER2 profileprovided within this specification.

Preferably, the lipolytic enzyme in accordance with the presentinvention comprises the GDSX (SEQ ID NO: 31) motif.

When aligned to either the pfam Pfam00657 consensus sequence (asdescribed in WO04/064987) and/or the L131 sequence herein disclosed (SEQID No 4)

-   -   i) The galactolipase/lipid acyl-transferase enzyme enzyme of the        invention, or for use in methods of the invention, has        preferably a GDSx (SEQ ID NO: 31) motif, more preferably a GDSY        (SEQ ID NO: 34) motif.    -   and/or    -   ii) The galactolipase/lipid acyl-transferase enzyme enzyme of        the invention, or for use in methods of the invention, has        preferably a GANDY (SEQ ID NO: 35) block, more preferably a        GANDY (SEQ ID NO: 35) block comprising amino GGNDx (SEQ ID NO:        36), more preferably GGNDA (SEQ ID NO: 37) or GGNDL (SEQ ID NO:        38).    -   and/or    -   iii) The enzyme of the invention, or for use in methods of the        invention, has preferable an HTP block.    -   and preferably    -   iv) The galactolipase/lipid acyl-transferase enzyme of the        invention, or for use in methods of the invention, has        preferably a GDSY (SEQ ID NO: 34) motif and a GANDY (SEQ ID        NO: 35) block comprising amino GGNDx (SEQ ID NO: 36), preferably        GGNDA (SEQ ID NO: 37) or GGNDL (SEQ ID NO: 38), and a HTP block        (conserved histadine).

In this regard, the inventors identified a homologous sequence toStreptomyces L131 which did not comprise a GDSX (SEQ ID NO: 31) motif:namely Novosphingobium aromaticivorans (NAL)

Novosphingobium\aromaticivorans\ GDSx (SEQ ID NO: 31) 284 aa

ZP_00094165 SEQ ID No. 10 1mgqvklfarr capvllalag lapaatvare aplaegaryv algssfaagp gvgpnapgsp 61ercgrgtlny phllaealkl dlvdatcsga tthhvlgpwn evppqidsvn gdtrlvtlti 121ggndvsfvgn ifaaacekma spdprcgkwr eiteeewqad eermrsivrq iharaplarv 181vvvdyitvlp psgtcaamai spdrlaqsrs aakrlarita rvareegasl lkfshisrrh 241hpcsakpwsn glsapaddgi pvhpnrlgha eaaaalvklv klmk / SEQ ID No. 11 1tgccggaact caagcggcgt ctagccgaac tcatgcccga aagcgcgtgg cactatcccg 61aagaccaggt ctcggacgcc agcgagcgcc tgatggccgc cgaaatcacg cgcgaacagc 121tctaccgcca gctccacgac gagctgccct atgacagtac cgtacgtccc gagaagtacc 181tccatcgcaa ggacggttcg atcgagatcc accagcagat cgtgattgcc cgcgagacac 241agcgtccgat cgtgctgggc aagggtggcg cgaagatcaa ggcgatcgga gaggccgcac 301gcaaggaact ttcgcaattg ctcgacacca aggtgcacct gttcctgcat gtgaaggtcg 361acgagcgctg ggccgacgcc aaggaaatct acgaggaaat cggcctcgaa tgggtcaagt 421gaagctcttc gcgcgccgct gcgccccagt acttctcgcc cttgccgggc tggctccggc 481ggctacggtc gcgcgggaag caccgctggc cgaaggcgcg cgttacgttg cgctgggaag 541ctccttcgcc gcaggtccgg gcgtggggcc caacgcgccc ggatcgcccg aacgctgcgg 601ccggggcacg ctcaactacc cgcacctgct cgccgaggcg ctcaagctcg atctcgtcga 661tgcgacctgc agcggcgcga cgacccacca cgtgctgggc ccctggaacg aggttccccc 721tcagatcgac agcgtgaatg gcgacacccg cctcgtcacc ctgaccatcg gcggaaacga 781tgtgtcgttc gtcggcaaca tcttcgccgc cgcttgcgag aagatggcgt cgcccgatcc 841gcgctgcggc aagtggcggg agatcaccga ggaagagtgg caggccgacg aggagcggat 901gcgctccatc gtacgccaga tccacgcccg cgcgcctctc gcccgggtgg tggtggtcga 961ttacatcacg gtcctgccgc catcaggcac ttgcgctgcc atggcgattt cgccggaccg 1021gctggcccag agccgcagcg ccgcgaaacg gcttgcccgg attaccgcac gggtcgcgcg 1081agaagagggt gcatcgctgc tcaagttctc gcatatctcg cgccggcacc atccatgctc 1141tgccaagccc tggagcaacg gcctttccgc cccggccgac gacggcatcc cggtccatcc 1201gaaccggctc ggacatgctg aagcggcagc ggcgctggtc aagcttgtga aattgatgaa 1261gtagctactg cactgatttc aaatagtatt gcctgtcagc tttccagccc ggattgttgc 1321agcgcaacag aaacttgtcc gtaatggatt gatggtttat gtcgctcgca aattgccgtc 1381gaagggaacg ggcgcgtcgc tcgttaacgt cctgggtgca gcagtgacgg agcgcgtgga 1441tgagtgatac tggcggtgtc atcggtgtac gcgccgccat tcccatgcct gtacgcgccg //

This enzyme comprises the sequence “GSSF” (SEQ ID NO: 39) as opposed toGDSX (SEQ ID NO: 31).

When tested it was found that this enzyme does not comprise glycolipaseactivity in accordance with the present invention.

Therefore, the GDSx (SEQ ID NO: 31) motif may be important whenattempting to identify other suitable galactolipases.

Notably, the enzyme from S. rimosus that has been purified andcharacterised biochemically and shows about 56% sequence homology toStreptomyces L131 (Abramić M., et al. (1999); Vujaklija D. et al.(2002)) is known to hydrolyse neutral lipids such as triolein ornitrophenyl esters of fatty. The enzyme from S. rimosus may alsohydrolyse galactolipase in accordance with the present invention.Similarly, two other Streptomyces species for which genome sequence datais available—S. coelicolor A2(3) and S. avermitilis may contain enzymeshaving galactolipase activity, for example (NP_(—)625998 andNP_(—)827753) are currently annotated in GenBank as “putative secretedhydrolases”.

Many other useful homologues of Streptomyces L131 galactolipase can beidentified by a similar approach. Suitable galactolipase/lipidacyl-transferase enzyme enzymes for use in the methods of the inventionmay be identified by alignment to the L131 sequence using Align X, theClustal W pairwise alignment algorithm of VectorNTI using defaultsettings.

Alternatively, suitable galactolipase for use in the methods of theinvention may be identified by alignment to the pfam Pfam00657 consensussequence (as described in WO04/064987).

FIG. 15 shows an sequence alignment of the L131 and homologues from S.avermitilis and T. fusca. FIG. 15 illustrates the conservation of theGDSx (SEQ ID NO: 31) motif (GDSY (SEQ ID NO: 34) in L131 and S.avermitilis and T. fusca), the GANDY (SEQ ID NO: 35) box, which iseither GGNDA (SEQ ID NO: 37) or GGNDL (SEQ ID NO: 38), and the HPT block(considered to be the conserved catalytic histadine). These threeconserved blocks are highlighted in FIG. 15.

When aligned to either the pfam Pfam00657 consensus sequence (asdescribed in WO041064987) and/or the L131 sequence herein disclosed (SEQID No 4) it is possible to identify three conserved regions, the GDSx(SEQ ID NO: 31) block, the GANDY (SEQ ID NO: 35) block and the HTP block(see WO041064987 for further details).

Example 10 Gene Cloning and Construction of Expression Vectors

Corynebacterium efficiens DSM 44549, Thermobifida fusca DSM 43792 andStreptomyces avermitilis DSM46492 were used for isolating the geneshomologous to the galactolipase gene of S. thermosacchari L131.

The strains accorded with a DSM number are deposited and publiclyavailable with Deutsche Sammlung von Mikroorganismen and ZellkulturenGmbH (DSM).

Escherichia coli strains XL-Blue MRF′, BL21(DE3) (Novagen) and S17-1(Simon R. et al., 1983), Bacillus subtilis BD170, Streptomyces lividansstrain 1326 (John Innes Centre), Corynebacterium glutamicum DSM20300were used as the hosts for heterologous expression. The strain ofAeromonas salmonicida (DSM 12609) was also used as an expression host.

S. thermosacchari L131, Citrobacter freundii P3-42 and Enterobacternimipressuralis P1-60 were isolated in our laboratory from naturalenvironment and taxonomically identified by 16S rRNA gene sequencing.

The following culture media were used in this study. LB (5 g/l yeastextract, 10 g/l tryptone, 10 g/l NaCl, pH 7.0), 2xYT (10 g/l NaCl, 10g/l yeast extract, 16 g/l tryptone) were used for cultivation of E. coliand other Gram-negative bacteria. Nutrient broth (3 g/l beef extract, 5g/l peptone, pH 7.0) was used for growing C. efficiens and N.aromaticivorans, YM-broth (3 g/l yeast extract, 3 g/l malt extract, 5g/l peptone, 10 g/l dextrose, pH 7.0) was used for cultivation of S.avermitilis, Medium 65 (4 g/l glucose, 4 g/l tryptone, 10 g/l maltextract, 2 g/l CaCO₃, pH 7.2) was used for T. fusca.

DNA Isolation.

Standard alkaline lysis procedure combined with Qiagen columnpurification method was used for plasmid isolation. One exception wasthe preparative isolation of plasmid DNA from Streptomyces. In thiscase, equilibrium centrifugation in CsCl gradient was used as the finalpurification step.

Methods for Introduction of DNA into Microbial Strains.

Both E. coli and C. glutamicum strains were transformed byelectroporation using 1 mm cuvettes and the following electroporationparameter settings: 1800V, 25° F., 200 μl B. subtilis BD170 wastransformed by “Paris” method based on natural competence (Harwood C. R.and Cutting S. M., 1990). Streptomyces lividans was transformed byprotoplast method (Kieser T. et al., 2000). DNA was introduced into A.salmonicida by conjugation with E. coli using filter mating method ofHarayama et al. (1980).

Construction of Rifampicin-Resistant Mutant of A. salmonicida.

About 10⁸ cells from overnight culture of A. salmonicida DSM12609 wereplated on a series of LB agar plates containing 5-30 mg/l rifampicin.The plates were irradiated by short wave UV light using SpectroLinkerXL-1500 device (Spectronics Corp. USA). The radiation dose was 4-6 J/M².The plates were incubated at 30° C. for 2 days. Several colonies growingon 30 mg/l rifampicin were selected and additionally tested on 50 mg/lrifampicin. One clone resistant to 50 mg/l rifampicin (named R1) waschosen for subsequent work.

Construction of E. coli Expression Vectors for L131 GalactolipaseHomologues.

The lipase gene of Streptomyces avermitilis was amplified by PCR usingchromosomal DNA as template and the two oligonucleotide primers oSAL-5(GGGAATTCCATATGAGACGTTCCCGAATTACG) (SEQ ID NO: 24) and oSAL-3(GCATGGATCCGGTGACCTGTGCGACGG) (SEQ ID NO: 25). For amplification oflipase genes of Thermobifida fusca and Corynebacterium effciens theoligonucleotide primers used were oTFL-5(GGGAATTCCATATGGGCAGCGGACCACGTG) (SEQ ID NO: 26) and oTFL-3(GCATGGATCCGACACGCACGGCTCAACG) (SEQ ID NO: 27), oCEL-5(GGGAATTCCATATGAGGACAACGGTCATCG) (SEQ ID NO: 28) and oCEL-3(GCATGGATCCGGCATCGGGCTCATCC) (SEQ ID NO: 29), respectively. The PCRproducts were digested with NdeI and BamHI and ligated with pET11 a(Novagen, USA) vector digested with the same restriction endonucleases.

L131 galactolipase expression vector for S. lividans was constructed asfollows. Plasmid pUC18(L131RX) that contains the 1.37 kb EcoRI-XbaIfragment of the original cloned DNA fragment carrying L131 lipase gene(pBK(L131)) was digested with EcoRI and ligated with EcoRI digestedpIJ487 (Kieser et al., 2000). This ligation leads to the formation ofthe two recombinant plasmids differing in relative orientation of pIJ487and pUC18(L131RX). For subsequent work a variant where lac promoter ofthe pUC18 is flanking the promoter-less neo^(R) gene of pIJ487 has beenselected based on restriction analysis. This construction was namedpRX487-5 (FIG. 11). Besides ampicillin resistance, this plasmid alsoconfers E. coli the resistance to at least 3 mg/l kanamycin. Theprotoplasts of S. lividans 1326 were transformed with 0.1-10 μg ofpRX487-5 to thiostreptone (1.2 mg/l) and kanamycin (5 mg/l) resistance.These transformants produced active galactolipase as judged by theDGDG-safranine indicator plate assay. The transformants were plated onSM plates (Kieser et al., 2000) supplemented with 5 mg/ml of kanamycinand allowed to sporulate. The resulting spores were used for inoculatingshake flask and fermentor cultures.

Construction of Expression Vectors for Corynebacterium glutamicum.

All expression vectors used in this work are based on the plasmid pCB5which is a shuttle vector carrying C. glutamicum replicon from plasmidpSR1 (Yoshihama et al., 1985) and ColE1 replicon from E. coli. Thepromoter that is used in this vector is derived from the cop1 geneencoding the major secreted protein of C. glutamicum-PS1. Enzymes wereexpressed from their native genes including unmodified signal peptides,e.g. T. fusca (FIG. 14).

Fermentation Conditions Fermentation of Lipase-Producing StreptomycesStrains.

In shake flasks, lipase-producing recombinant S. lividans strains weregrown in a medium containing (per litre) 10 g peptone, 5 g yeastextract, 2 g K₂HPO₄ and 10 g glucose (pH 7.0) supplemented withappropriate antibiotics: thiostreptone was used at 1.2 mg/l, kanamycinat 20 mg/l, chloramphenicol at 1.5 mg/l and erythromycin at 1.5 mg/l.Spore suspensions produced by growing the transformants on SM plateswere used to start the cultivations.

For fed-batch fermentations, Braun Biostat E fermentor (10 l) was used.The initial medium (7 l), contained (per litre): peptone 20 g, yeastextract, 10 g, glucose 20 g and appropriate antibiotics as describedabove (except for thiostreptone, which was not used in 10 l cultures).The cultivation was conducted at 30° C., constant 10 l/min aeration and600 rpm stirring rate. Inocula (2×250 ml per fermentation) were grown in2 l Erlenmeyer flasks as described in the previous paragraph. Thefermentation was carried out in batch mode for 18-20 h after which time,a solution containing 30% glucose and 12.5% peptone was fed to thefermentor culture at a rate of 0.5 ml/min. Samples (30 ml) of theculture were withdrawn aseptically twice a day.

Fermentation of Recombinant C. glutamicum Strains.

Shake-flask cultures of C. glutamicum were grown in LB containing 50mg/l kanamycin at 30° C. and 200 rpm agitation rate.

Fermentation of Recombinant A. salmonicida Strains.

In shake flasks, the recombinant A. salmonicida strains were cultivatedin 2xYT medium supplemented with streptomycin and kanamycin (at 25mg/l). To induce tac promoter, IPTG (1-5 mM) or lactose (1-10%) wereadded to the growth medium.

Two sets of conditions for production of recombinant acyl-transferase inA. salmonicida were tested at fermentor scale. In the first experiment,the initial medium (7 l) was 2xYT supplemented with 2% glucose, 50 mg/lof kanamycin and 50 mg/l of streptomycin and the feeding solution (3 l)contained 25% glucose, 10% tryptone and 5% yeast extract, 100 mg/l ofboth kanamycin and streptomycin. Cultivation was carried out at 10 l/minaeration, 600 rpm stirring rate and 28° C. The pH was adjusted to 7.5 by25% NH₃ and 10% phosphoric acid. The fermentor was inoculated with 0.5 lof overnight culture of A. salmonicida and grown in batch mode for 24 h.At this point IPTG was added to 5 mM and the feeding was started at arate of 50 ml/h.

In the second experiment, the initial medium was modified bysubstituting glucose with lactose. Feeding solution was 2 l of 20%lactose. The fermentation temperature was increased to 30° C. and the pHof the culture medium decreased to 7.0. Inoculation was done as in thefirst experiment and the feeding (100 ml/h) was started after 20 h ofcultivation in the initial medium

Enzyme Assays Safranine Plate Screening Method.

In safranine plate screening the bottom layer contained culturemedium+additive, 1.5% agarose and 0.002% safranine (0.2% stock solutionin water, sterile filtered) and the top layer 0.7% agarose, 1% DGDG and0.002% safranine.

Determination of Galactolipase Activity (Glycolipase Activity Assay(GLU-7)): Substrate:

0.6% digalactosyldiglyceride (Sigma D 4651), 0.4% Triton-X 100 (SigmaX-100) and 5 mM CaCl₂ was dissolved in 0.05M HEPES buffer pH 7.

Assay Procedure:

400 μL substrate was added to an 1.5 mL Eppendorf tube and placed in anEppendorf Thermomixer at 37° C. for 5 minutes. At time t=0 min, 50 μLenzyme solution was added. Also a blank with water instead of enzyme wasanalyzed. The sample was mixed at 10×100 rpm in an Eppendorf Thermomixerat 37° C. for 10 minutes. At time t=10 min the Eppendorf tube was placedin another thermomixer at 99° C. for 10 minutes to stop the reaction.

Free fatty acid in the samples was analyzed by using the NEFA C kit fromWAKO GmbH.

Enzyme activity GLU at pH 7 was calculated as micromole fatty acidproduced per minute under assay conditions

Determination of Phospholipase Activity (Phospholipase Activity Assay(PLU-7)): Substrate

0.6% L-α Phosphatidylcholine 95% Plant (Avanti #441601), 0.4% Triton-X100 (Sigma X-100) and 5 mM CaCl₂ was dispersed in 0.05M HEPES buffer pH7.

Assay Procedure:

400 μL substrate was added to an 1.5 mL Eppendorf tube and placed in anEppendorf Thermomixer at 37° C. for 5 minutes. At time t=0 min, 50 μLenzyme solution was added. Also a blank with water instead of enzyme wasanalyzed. The sample was mixed at 10×100 rpm in an Eppendorf Thermomixerat 37° C. for 10 minutes. At time t=10 min the Eppendorf tube was placedin another thermomixer at 99° C. for 10 minutes to stop the reaction.

Free fatty acid in the samples was analyzed by using the NEFA C kit fromWAKO GmbH.

Enzyme activity PLU-7 at pH 7 was calculated as micromole fatty acidproduced per minute under assay conditions.

Spectrophotometric Assay with p-nitrophenyl Palmitate (pNPP).

Lipase activity was measured with a spectrophotometric assay at 30° C.with pNPP as substrate, by using 50 mM Tris-Maleate buffer (pH 6.5) with0.4% Triton X-100 and 0.1% gum Arabic. The substrate stock solution (100mM) was prepared in dioxane. The kinetic measurement was started byaddition of enzyme to the reaction mixture. To evaluate the initialhydrolytic activity, the increase in absorption at 410 nm was followedwith Spectramax plate reader every 20 s for 20 min. One unit of lipaseactivity was defined as the amount of enzyme that liberated 1 μmol ofp-nitrophenol per min. The activity toward other p-NP esters wasmeasured in the same manner, by using 1 mM each substrate. (Abramic M.et al. (1999))

Determination of Effects of pH and Temperature on Lipase Activity.

For the determination of the effect of pH on enzymatic activity, it wasmeasured over a range of pH 2-10 by using the galactolipase activityassay except that the buffers used in the experiment were as follows: pH2-3.5 Glycine-HCl; pH 4-5 NaOAc; pH 5.5-7.5 Tris-Maleate; pH 7.5-9Tris-HCl; pH 10 CAPS.

The effect of temperature on galactolipase stability was determined byincubating aliquots of enzyme for 20 min at various temperatures (22°C.-90° C.) following incubation on ice for 60 min. Residual activity wasanalysed by galactolipase activity assay.

For detection of optimal temperature for galactolipase activity, theusual assay mixture was equilibrated at the required temperature (therange 20° C.-70° C.) and 2 or 4 μl of enzyme was added to start thereaction. The activity was analysed by galactolipase activity assay, butusing a shorter period of time (20 min).

Example 11 Characterisation of Galactolipase Candidates fromBiodiversity Study

The sequence of Streptomyces thermosacchari L131 galactolipase offersfor the possibility for in silico identification of new family IIgalactolipases.

Many other useful homologues of Streptomyces L131 galactolipase can beidentified, for example, “hypothetical protein” from Thermobifida fusca(ZP_(—)00058717) and “hypothetical protein” from Corynebacteriumefficiens (NP_(—)738716).

We cloned and expressed 3 homologues of Streptomyces L131 galactolipase:the genes of Streptomyces avermitilis (SAL), Thermobifida fusca (TFL),and Corynebacterium efficiens (CEL). All genes were expressed in E. coliby using pET expression system. The recombinant E. coli strains werefirst analysed using DGDG—indicator plates with safranine and theenzymes of S. avermitilis, T. fusca and C. efficiens were found to havegalactolipase activity.

The enzymes showing galactolipase activity were further examined.Substrate specificities of those galactolipase candidates were studied(FIG. 13). The activity of candidate enzymes towards DGDG, lecithin,olive oil, nitrophenyl butyrate, nitrophenyl decanoate (NP-D) andnitrophenyl palmitate was tested. The enzymes were found to have verydifferent substrate specificity profiles. Acyl-transferase activity wastested in an assay using NP-D as substrate and quantifying both therelease of nitrophenol and free fatty acids by NEFA kit. Preliminarydata suggests that at least the enzyme from Thermobifida fusca hastransferase activity towards glycerol and glucose.

Thermo-stability of galactolipase candidates was tested. It was foundthat the Corynebacterium efficiens enzyme was the most thermostablewhile the enzyme of Streptomyces avermitilis was the mostthermo-sensitive.

Example 12 Streptomyces thermosacchari L131 Degumming Trial

A phospholipase from Streptomyces thermosacchari L131 was tested incrude soya oil.

Materials and Methods

K371: Streptomyces thermosacchari L131 enzyme expressed in S. lividansfreeze dried on starch.

(Activity: 108 PLU-7/g).

Lecitase Ultra (#3108) from Novozymes, Denmark

Cholesterolester, Fluka 26950

Plant Sterol Generol 122 N from Henkel, GermanyCrude soya oil from The Solae Company, Aarhus Denmark

Lecithin: L-α Phosphatidylcholine 95% Plant (Avanti #441601)Phospholipase Activity

The phospholipase assay was the same as that used in Example 10.

HPTLC Applicator: Automatic TLC Sampler 4, CAMAG

HPTLC plate: 20×10 cm, Merck no. 1.05641. Activated 30 minutes at 160°C. before use.Application: 1 μl of a 8% solution of oil in buffer was applied to theHPTLC plate using Automatic TLC applicator.Running buffer 4: Chloroform:Methanol:Water 75:25:4Running buffer 5: P-ether: Methyl-tert-butyl ketone: Acetic acid 70:30:1

Application/Elution Time:

Running buffer 4: 20 minRunning buffer 5: 10 min

TLC Development

The plate was dried in an oven for 10 minutes at 160° C., cooled, anddipped into 6% cupri acetate in 16% H₃PO₄. Dried additionally 10 minutesat 160° C. and evaluated directly.

Degumming Experiment

Streptomyces thermosacchari L131 (K371) was used for degumming studiesin the formulations shown in table 4.

The samples were placed at 40° C. for 18 hours with agitation, afterwhich time a sample was collected for HPTLC analysis by dissolving thesample in Chloroform:Methanol 2:1

TABLE 4 Degumming of crude soya oil with Streptomyces thermosacchariL131 And Lecitase Ultra ™ Lane 1 2 3 4 5 6 Crude soya oil % 99 99 98 9799.7 99 K371, 10% in water % 1 2 3 Lecitase Ultra ™ #3108, 1% in % 0.30.3 water Water % 1 0 0 0 0.7

The results from the HPTLC analysis are shown in FIG. 16 and FIG. 17.

FIG. 16 shows TLC plate (Buffer 4) of reaction products from enzymetreatment of crude soya oil samples according to table 4. As referenced,phosphatidylcholine (PC) was also analysed. Phosphatydylethanolamine(PE) and lysophosphatidylcholine (LPC) are also indicated.

The TLC results in FIG. 16 clearly show that phosphatidylcholine wascompletely removed by adding Streptomyces thermosacchari L131 to theoil. Only the lowest dosage (lane 2) did not completely hydrolyse thephospholipids. Lecitase Ultra™ also hydrolysed the phospholipids in theoil when 5% water was available (Lane 6) but without adding extra water(Lane 5) only part of the phospholipids were hydrolysed.

FIG. 17 shows TLC (Buffer 5) of reaction products from enzyme treatmentof crude soya oil samples according to table 4. As referenced,cholesterolester, monoglyceride, diglyceride, triglyceride and plantsterol. Free fatty acid (FFA) is also indicated

The results shown in FIG. 17 indicate that the hydrolysis ofphospholipids is coincident with the formation of free fatty acid.

CONCLUSION

The results confirm that Streptomyces thermosacchari L131 effectivelyhydrolyses phospholipids in crude soya oil and is a suitable alternativeenzyme for degumming of plant oils.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

REFERENCES

-   Abramic M., Lescic I., Korica T., Vitale L., Saenger W., Pigac J.    Purification and properties of extracellular lipase from    Streptomyces rimosus. Enzyme and Microbial Technology 25 522-529    (1999)-   Harayama S., Masataka T., Iino T. High-frequency mobilisation of the    chromosome of Escherichia coli by a mutant of plasmid RP4    temperature sensitive for maintenance. Mol. Gen. Genet. 180, 47-56    (1980).-   Harwood C. R. and Cutting S. M. Molecular biological methods for    Bacillus. John Wiley & Sons Ltd., West Sussex, England (1990)-   Kieser T., Bibb M. J., Buttner M. J., Chater K. F., Hopwood D. A.    Practical Streptomyces genetics. The John Innes Foundation, Crowes,    Norwich, England (2000)-   Vujaklija D., Schroder W., Abramic M., Zou P., Lescic I., Franke P.,    Pigac J. A novel streptomycete lipase: cloning, sequencing and    high-level expression of the Streptomyces rimosus GDS(L)-lipase    gene. Arch. Microbiol. 178, 124-130 (2002)-   Yoshihama M, Higashiro K, Rao E A, Akedo M, Shanabruch W G,    Follettie M T, Walker G C, Sinskey A J. Cloning vector system for    Corynebacterium glutamicum. J. Bacteriol. 162 (2):591-597 (1985).

The invention will now be further described by the following numberedparagraphs:

1. A lipolytic enzyme capable of hydrolysing at least a galactolipidand/or capable of transferring an acyl group from a galactolipid to oneor more acyl acceptor substrates, wherein the enzyme is obtainable fromStreptomyces species.2. A lipolytic enzyme capable of hydrolysing polar lipids and/or capableof transferring an acyl group from a polar lipid to one or more acylacceptor substrates, wherein the enzyme is encoded by a nucleic acidselected from the group consisting of: a) a nucleic acid comprising anucleotide sequence shown in SEQ ID No. 3; b) a nucleic acid which isrelated to the nucleotide sequence of SEQ ID No. 3 by the degenerationof the genetic code; and c) a nucleic acid comprising a nucleotidesequence which has at least 70% identity with the nucleotide sequenceshown in SEQ ID No. 3.3. A lipolytic enzyme according to paragraph 1 or paragraph 2 comprisingan amino acid sequence as shown in SEQ ID No. 4 or an amino acidsequence which has at least 60% identity thereto.4. A lipolytic enzyme obtainable from the Streptomyces strains L130 orL131 deposited under accession numbers NCIMB 41226 and NCIMB 41227,respectively.5. A lipolytic enzyme according to any one or more of paragraphs 2-4wherein the enzyme is capable of hydrolysing at least a galactolipidand/or is capable of transferring an acyl group from a galactolipid toone or more acyl acceptor substrates.6. A lipolytic enzyme according to paragraph 1 or paragraph 5 whereinthe enzyme is capable of hydrolysing a further polar lipid.7. A lipolytic enzyme according to paragraph 6 wherein the polar lipidis a phospholipid.8. A lipolytic enzyme according to paragraph 1 or paragraph 5 whereinthe lipolytic enzyme is capable of transferring an acyl group from apolar lipid to one or more of the following acyl acceptor substrates: asterol, a stanol, a carbohydrate, a protein or subunits thereof, or aglycerol.9. A lipolytic enzyme according to any one of the preceding paragraphswherein the enzyme is a wild-type enzyme.10. A nucleic acid encoding a lipolytic enzyme comprising an amino acidsequence as shown in SEQ ID No. 4 or an amino acid sequence which has atleast 60% identity therewith.11. A nucleic acid encoding a lipolytic enzyme, which nucleic acid isselected from the group consisting of: a) a nucleic acid comprising anucleotide sequence shown in SEQ ID No. 3; b) a nucleic acid which isrelated to the nucleotide sequence of SEQ ID No. 3 by the degenerationof the genetic code; and c) a nucleic acid comprising a nucleotidesequence which has at least 70% identity with the nucleotide sequenceshown in SEQ ID No. 3.12. Use of a lipolytic enzyme according to any one of paragraphs 1-9 ina process of preparing a lyso-glycolipid, for example digalactosylmonoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG) by treatmentof a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monogalactosyldiglyceride (MGDG)) with the lipolytic enzyme according to the presentinvention to produce the partial hydrolysis product, i.e. thelyso-glycolipid.13. Use of a lipolytic enzyme according to any one of paragraphs 1-9 ina process of preparing a lyso-phospholipid, for example lysolecithin, bytreatment of a phospholipid (e.g. lecithin) with the enzyme according tothe present invention to produce a partial hydrolysis product, i.e. alyso-phospholipid.14. Use of a lipolytic enzyme according to any one of paragraphs 1-9 ina process of enzymatic degumming of vegetable or edible oil, comprisingtreating said edible or vegetable oil with said lipolytic enzyme so asto hydrolyse a major part of the polar lipids.15. Use of a lipolytic enzyme according to any one of paragraphs 1-9 ina process of comprising treatment of a phospholipid so as to hydrolysefatty acyl groups.16. Use of a lipolytic enzyme according to any one of paragraphs 1-9 ina process of bioconversion of polar lipids to make high value products,wherein said lipolytic is capable of hydrolysing said polar lipids.17. Use according to paragraph 16 wherein said high value products areone or more of the following: a carbohydrate ester, a protein ester, aprotein subunit ester and a hydroxy acid ester.18. A method of preparing a foodstuff the method comprising admixing thelipolytic enzyme according to any one of paragraphs 1-9 to one or moreingredients of the foodstuff.19. A method according to paragraph 14 wherein the foodstuff is selectedfrom one or more of the following: eggs, egg-based products, includingbut not limited to mayonnaise, salad dressings, sauces, ice creams, eggpowder, modified egg yolk and products made therefrom; baked goods,including breads, cakes, sweet dough products, laminated doughs, liquidbatters, muffins, doughnuts, biscuits, crackers and cookies;confectionery, including chocolate, candies, caramels, halawa, gums,including sugar free and sugar sweetened gums, bubble gum, soft bubblegum, chewing gum and puddings; frozen products including sorbets,preferably frozen dairy products, including ice cream and ice milk;dairy products, including cheese, butter, milk, coffee cream, whippedcream, custard cream, milk drinks and yoghurts; mousses, whippedvegetable creams, meat products, including processed meat products;edible oils and fats, aerated and non-aerated whipped products,oil-in-water emulsions, water-in-oil emulsions, margarine, shorteningand spreads including low fat and very low fat spreads; dressings,mayonnaise, dips, cream based sauces, cream based soups, beverages,spice emulsions and sauces.20. A method according to paragraph 19 wherein said foodstuff is a dairyproduct.21. A method according to paragraph 19 wherein said foodstuff is an eggor egg-based product.22. A method according to paragraph 19 wherein said foodstuff is a dairyproduct.23. A method of preparing a lysoglycolipid comprising treating asubstrate comprising a glycolipid with at least one lipolytic enzyme toproduce said lysoglycolipid, wherein said lipolytic enzyme hasglycolipase activity and wherein said lipolytic enzyme is obtainablefrom one of the following genera: Streptomyces, Corynebacterium andThermobifida.24. A method of preparing a lysophospholipid comprising treating asubstrate comprising a phospholipid with at least one lipolytic enzymeto produce said lysophospholipid, wherein said lipolytic enzyme hasphospholipase activity and wherein said lipolytic enzyme is obtainablefrom one of the following genera: Streptomyces, Corynebacterium andThermobifida.25. A method of enzymatic degumming of vegetable or edible oil,comprising treating said edible or vegetable oil with a lipolytic enzymeobtainable from one of the following genera: Streptomyces,Corynebacterium and Thermobifida capable of hydrolysing a major part ofthe polar lipids.26. A method of bioconversion of polar lipids to make high valueproducts comprising treating said polar lipids with a lipolytic enzymeobtainable from one of the following genera: Streptomyces,Corynebacterium and Thermobifida to produce said high value products,wherein said lipolytic enzyme is capable of hydrolysing said polarlipids.27. A method according to paragraph 26 wherein said high value productsare one or more of the following: a carbohydrate ester, a protein ester,a protein subunit ester and a hydroxy acid ester.28. A method of preparing a foodstuff comprising admixing at least onelipolytic enzyme with one or more ingredients of a foodstuff whereinsaid lipolytic enzyme is capable of hydrolysing a glycolipid and/or aphospholipid present in or as at least one of said ingredients andwherein said lipolytic enzyme is obtainable from one of the followinggenera: Streptomyces, Corynebacterium and Thermobifida.29. A method according to any one of paragraphs 23 to 28 wherein saidlipolytic enzyme is capable of transferring an acyl group from aglycolipid to one or more acyl acceptor substrates.30. A method according to any one of paragraphs 23 to 28 wherein saidlipolytic enzyme comprises an amino acid sequence shown as SEQ ID Nos 5,7, 8, 12, 14 or 16 or an amino acid sequence having at least 70%identity therewith or comprises a nucleotide sequence shown as SEQ ID No6, 9, 13, 15 or 17 or a nucleotide sequence which has at least 70%identity therewith.31. A method according to paragraph 30 wherein said lipolytic enzymecomprises an amino acid sequence shown as SEQ ID Nos 5, 7, or 16 or anamino acid sequence having at least 70% identity therewith.32. A method according to paragraphs 30 wherein said lipolytic enzymecomprises an amino acid sequence shown as SEQ ID No. 8 or an amino acidsequence having at least 70% identity therewith.33. A method according to paragraphs 30 wherein said lipolytic enzymecomprises an amino acid sequence shown as SEQ ID Nos 12 or 14 or anamino acid sequence having at least 80% identity therewith.34. A method according to paragraph 28 wherein said foodstuff isselected from one or more of the following: eggs, egg-based products,including but not limited to mayonnaise, salad dressings, sauces, icecreams, egg powder, modified egg yolk and products made therefrom; bakedgoods, including breads, cakes, sweet dough products, laminated doughs,liquid batters, muffins, doughnuts, biscuits, crackers and cookies;confectionery, including chocolate, candies, caramels, halawa, gums,including sugar free and sugar sweetened gums, bubble gum, soft bubblegum, chewing gum and puddings; frozen products including sorbets,preferably frozen dairy products, including ice cream and ice milk;dairy products, including cheese, butter, milk, coffee cream, whippedcream, custard cream, milk drinks and yoghurts; mousses, whippedvegetable creams, meat products, including processed meat products;edible oils and fats, aerated and non-aerated whipped products,oil-in-water emulsions, water-in-oil emulsions, margarine, shorteningand spreads including low fat and very low fat spreads; dressings,mayonnaise, dips, cream based sauces, cream based soups, beverages,spice emulsions and sauces.35. A method according to paragraph 34 wherein said foodstuff is a bakedproduct and at least one of said ingredients is a dough.36. A method according to paragraph 34 wherein said foodstuff is an eggor egg-based product.37. A method according to paragraph 34 wherein said foodstuff is a dairyproduct.38. Use of a lipolytic enzyme in a substrate for preparing alysoglycolipid wherein said lipolytic enzyme has glycolipase activityand wherein said lipolytic enzyme is obtainable from one of thefollowing: Streptomyces, Corynebacterium and Thermobifida.39. Use of a lipolytic enzyme in a substrate for preparing alysophospholipid wherein said lipolytic enzyme has phospholipaseactivity and wherein said lipolytic enzyme is obtainable from one of thefollowing: Streptomyces, Corynebacterium and Thermobifida.40. Use of a lipolytic enzyme obtainable from one of the followinggenera: Streptomyces, Corynebacterium and Thermobifida for enzymaticdegumming of vegetable or edible oil so as to hydrolyse a major part ofthe polar lipids.41. Use of a lipolytic enzyme obtainable from one of the followinggenera: Streptomyces, Corynebacterium and Thermobifida in a processcomprising treatment of a phospholipid so as to hydrolyse fatty acylgroups.42. Use of a lipolytic enzyme in the bioconversion of polar lipids tomake high value products, wherein said lipolytic enzyme is capable ofhydrolysing said polar lipids and wherein said lipolytic enzymes isobtainable from one of the following genera: Streptomyces,Corynebacterium and Thermobifida.43. Use according to paragraph 42 wherein said high value products areone or more of the following: a carbohydrate ester, a protein ester, aprotein subunit ester and a hydroxy acid ester.44. Use of a lipolytic enzyme obtainable from one of the followinggenera: Streptomyces, Corynebacterium and Thermobifida in thepreparation of a foodstuff, wherein said lipolytic enzyme is capable ofhydrolysing a glycolipid and/or a phospholipid.45. Use according to paragraph 38 to 44 wherein said lipolytic enzyme iscapable of transferring an acyl group from a glycolipid to one or moreacyl acceptor substrates.46. Use according to any one of paragraphs 38 to 44 wherein saidlipolytic enzyme comprises an amino acid sequence as shown in any one ofSEQ ID Nos 5, 7, 8, 12, 14 or 16 or an amino acid sequence having atleast 70% identity therewith or comprises a nucleotide sequence shown asSEQ ID No 6, 9, 13, 15 or 17 or a nucleotide sequence which has at least70% identity therewith.47. Use according to paragraph 46 wherein said lipolytic enzymecomprises an amino acid sequence as shown in any one of SEQ ID Nos 5, 7,or 16 or an amino acid sequence having at least 70% identity therewith.48. Use according to paragraphs 46 wherein said lipolytic enzymecomprises an amino acid sequence shown as SEQ ID No 8 or an amino acidsequence having at least 70% identity therewith.49. Use according to paragraphs 46 wherein said lipolytic enzymecomprises an amino acid sequence shown as SEQ ID Nos 12 or 14 or anamino acid sequence having at least 80% identity therewith.

50. Use according to any one of paragraph 38 wherein said lysoglycolipidis DGMG or MGMG.

51. Use according to paragraph 44 wherein said foodstuff is a dairyproduct.52. Use according to paragraph 51 wherein said foodstuff is an egg or anegg-based product and wherein said lipolytic enzyme is capable oftransferring an acyl group to one or more acyl acceptor substrates toreduce the one or more of the following detrimental effects: off-odoursand/or off-flavours and/or soapy tastes.53. Use according to paragraph 51 wherein said foodstuff is a bakedproduct.54. Use according to paragraph 38 wherein said substrate is an edibleoil.55. A lipolytic enzyme as hereinbefore described with reference to theaccompanying description and figures.56. A method as hereinbefore described with reference to theaccompanying description and the figures.57. A use as hereinbefore described with reference to the accompanyingdescription and figures.

1-19. (canceled)
 20. A method of preparing a lysoglycolipid comprisingtreating a substrate comprising a glycolipid with at least one lipolyticenzyme to produce said lysoglycolipid, wherein said lipolytic enzyme hasglycolipase activity and wherein said lipolytic enzyme is obtainablefrom the genus Thermobifida.
 21. A method of preparing alysophospholipid comprising treating a substrate comprising aphospholipid with at least one lipolytic enzyme to produce saidlysophospholipid, wherein said lipolytic enzyme has phospholipaseactivity and wherein said lipolytic enzyme is obtainable from the genusThermobifida.
 22. A method of enzymatic degumming of vegetable or edibleoil, comprising treating said edible or vegetable oil with a lipolyticenzyme obtainable from the genus Thermobifida capable of hydrolysing amajor part of the polar lipids.
 23. A method of bioconversion of polarlipids to make high value products comprising treating said polar lipidswith a lipolytic enzyme obtainable from the genus Thermobifida toproduce said high value products, wherein said lipolytic enzyme iscapable of hydrolysing said polar lipids.
 24. A method according toclaim 23 wherein said high value products are one or more of thefollowing: a carbohydrate ester, a protein ester, a protein subunitester and a hydroxy acid ester.
 25. A method of preparing a foodstuffcomprising admixing at least one lipolytic enzyme with one or moreingredients of a foodstuff wherein said lipolytic enzyme is capable ofhydrolysing a glycolipid and/or a phospholipid present in or as at leastone of said ingredients and wherein said lipolytic enzyme is obtainablefrom the genus Thermobifida.
 26. A method according to any one of claims20 to 25 wherein said lipolytic enzyme is capable of transferring anacyl group from a glycolipid to one or more acyl acceptor substrates.27. A method according to any one of claims 20 to 25 wherein saidlipolytic enzyme comprises an amino acid sequence shown as SEQ. ID. No.5, 7 or 16 or an amino acid sequence having at least 70% identitytherewith or comprises a nucleotide sequence shown as SEQ ID No. 6 or 17or a nucleotide sequence which has at least 70% identity therewith. 28.A method according to any one of claims 20 to 25, wherein said lipolyticenzyme comprises an amino acid sequence shown as SEQ ID Nos 5, 7, or 16or an amino acid sequence having at least 70% identity therewith. 29-30.(canceled)
 31. A method according to claim 25 wherein said foodstuff isselected from one or more of the following: eggs, egg-based products,including but not limited to mayonnaise, salad dressings, sauces, icecreams, egg powder, modified egg yolk and products made therefrom; bakedgoods, including breads, cakes, sweet dough products, laminated doughs,liquid batters, muffins, doughnuts, biscuits, crackers and cookies;confectionery, including chocolate, candies, caramels, halawa, gums,including sugar free and sugar sweetened gums, bubble gum, soft bubblegum, chewing gum and puddings; frozen products including sorbets,preferably frozen dairy products, including ice cream and ice milk;dairy products, including cheese, butter, milk, coffee cream, whippedcream, custard cream, milk drinks and yoghurts; mousses, whippedvegetable creams, meat products, including processed meat products;edible oils and fats, aerated and non-aerated whipped products,oil-in-water emulsions, water-in-oil emulsions, margarine, shorteningand spreads including low fat and very low fat spreads; dressings,mayonnaise, dips, cream based sauces, cream based soups, beverages,spice emulsions and sauces.
 32. A method according to claim 31 whereinsaid foodstuff is a baked product and at least one of said ingredientsis a dough.
 33. A method according to claim 31 wherein said foodstuff isan egg or egg-based product.
 34. A method according to claim 32 whereinsaid foodstuff is a dairy product. 35-37. (canceled)
 38. A method ofhydrolyzing fatty acyl groups in a phospholipid comprising contactingthe phospholid with a lipolytic enzyme obtainable from Thermobifida,wherein the lipolytic enzyme hydrolizes the fatty acyl groups in aphospholipid. 39-46. (canceled)
 47. The method according to claim 20wherein said lysoglycolipid is DGMG or MGMG.
 48. The method according toclaim 25 wherein said foodstuff is a dairy product.
 49. The methodaccording to claim 48 wherein said foodstuff is an egg or an egg-basedproduct and wherein said lipolytic enzyme is capable of transferring anacyl group to one or more acyl acceptor substrates to reduce the one ormore of the following detrimental effects: off-odours and/oroff-flavours and/or soapy tastes.
 50. The method according to claim 48wherein said foodstuff is a baked product.
 51. The method according toclaim 20 wherein said substrate is an edible oil.